Device and method for in vivo detection of clots within circulatory vessels

ABSTRACT

A device and method of using the device to detect the presence and composition of clots and other target objects in a circulatory vessel of a living subject is described. In particular, devices and methods of detecting the presence and composition of clots and other target objects in a circulatory vessel of a living subject using in vivo photoacoustic flow cytometry techniques is described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/496,995 filed Oct. 8, 2021 which is a continuation of U.S. patentapplication Ser. No. 15/240,712 filed Aug. 18, 2016 which is adivisional of U.S. patent application Ser. No. 13/253,767, entitled“Device and Method for In Vivo Detection of Clots Within CirculatoryVessels” filed on Oct. 5, 2011, which is a continuation-in-part of U.S.patent application Ser. No. 12/945,576, entitled “Device and Method forIn Vivo Noninvasive Magnetic Manipulation of Circulating Objects inBioflows” filed on Nov. 12, 2010, which is a continuation-in-part ofU.S. patent application Ser. No. 12/334,217, entitled “Device and Methodfor In Vivo Flow Cytometry Using the Detection of Photoacoustic Waves”filed on Dec. 12, 2008, all of which are hereby incorporated byreference in their entireties. U.S. patent application Ser. No.12/334,217 claims priority from U.S. provisional patent application Ser.No. 61/013,543, entitled “Device and Method for In Vivo Flow CytometryUsing the Detection of Photoacoustic Waves” filed on Dec. 13, 2007.

GOVERNMENTAL RIGHTS IN THE INVENTION

This invention was made with government support under grant numbers R01EB000873, R21 EB005123, R01 CA131164, R21 CA139373, and R01 EB009230awarded by the National Institutes of Health as well as grant numberDBI-0852737 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This application relates to a device and methods of using the device todetect the presence and composition of clots and other target objects ina circulatory vessel of a living subject. In particular, thisapplication relates to devices and methods of detecting the presence andcomposition of clots and other target objects in a circulatory vessel ofa living subject using in vivo photoacoustic flow cytometry techniques.

BACKGROUND

Despite progress in the diagnosis and treatment of cardiovasculardiseases, heart attack and stroke remain among the leading causes ofdeath. One common risk factor for cardiovascular diseases is thepresence of circulating clots. These circulating clots (also known asthrombi) may unexpectedly block blood vessels, preventing delivery ofoxygen-rich blood to vital organs, such as the heart and brain, andpotentially causing unexpected acute events, including strokes and heartattacks. Although these acute events are typically caused by relativelylarge clots, victims of acute coronary thrombosis may harbor manymicrothrombi in vessels as small as 120 μm in diameter or less. Atpresent, no clinically relevant method has been developed for the earlydetection of circulating clots, despite their clinical significance asprognostic markers for incipient strokes and heart attacks, as well asthe potential for prevention of acute clot-related events throughwell-timed therapeutic clot elimination.

Existing ex vivo methods of detecting clots typically involve invasiveextraction of blood samples and time-consuming analysis techniques. Thetemporal resolution of existing ex vivo methods is limited by thediscrete time-point nature of drawing blood samples. As a result, theeffectiveness of existing ex vivo methods is limited for monitoring thedevelopment of clots over time. Further, the sensitivity of existing exvivo methods is relatively poor due to the difficulty of obtaining bloodsamples from clinically relevant sites, such as the carotid artery,limiting the analysis to relatively small-volume blood samples.

Many of the limitations of existing ex vivo methods may be overcomeusing the assessment of larger blood volumes in vivo. However, existingnon-invasive diagnostic techniques suitable for in vivo assessments suchas MRI, PET, ultrasound, and optical imaging are only capable ofdetecting fixed clots or slowly moving large clots in circulation.Existing fluorescent imaging techniques have been used to detect rollingclots in experimental models of colitis, and to assess the heterogeneityof adhered clots. However, the translation of existing fluorescentimaging methods from experimental models to in vivo use in humans may beproblematic due to the toxicity of the fluorescent labels used influorescent imaging, as well as the challenge of detecting clots againsta strong in vivo autofluorescent background. Other visualizationtechniques, such as pulse Doppler ultrasound, may be of limited use forclot screening due to the complexity of the technique, the difficulty ofclot recognition within the surrounding blood and tissues, andmeasurement artifacts related to air bubbles.

Photoacoustic (PA) imaging is a technique based on the detection oflaser-induced thermoelastic acoustic waves. PA imaging provides greaterdetection sensitivity and spatial resolution of target objects, such ascells and biomarker compounds, within tissues as deep as 3-5 cm comparedto other optical visualization techniques, such as fluorescent imaging.As applied to vein thrombosis staging, PA imaging techniques have beenused to detect stationary thrombus phantoms in vitro. However, PAdetection of circulating clots in vivo using existing techniques islimited by the slow signal acquisition algorithms currently in use.

A need exists for a device and method for detecting circulating clots invivo, continuously, and non-invasively with high detection sensitivityand spatial resolution. Such a device and method would make possible thedetection of clots ranging in size from microclots to larger,slower-moving clots over extended periods. The increased resolution mayfurther allow for the early detection of circulating microclots,facilitating the use of microclots as biomarkers for the earlyprediction of incipient acute events such as heart attacks or strokes.The early detection of microclots and the ability to monitor thedevelopment of clots over extended periods may better inform decisionssuch as the timing of therapeutic clot elimination treatments, as wellas monitor the effectiveness of such treatments.

SUMMARY

Aspects of the present invention provide a method for detecting a clotproperty within a circulatory vessel of a living organism. The methodincludes pulsing a clot within the circulatory vessel with at least onepulse of laser energy at a first pulse wavelength. The first pulsewavelength induces a photoacoustic signal from the clot that is lower inmagnitude than the photoacoustic signal induced from surrounding redblood cells. The method also includes obtaining a photoacoustic patternemitted by the clot induced by at least one pulse of laser energy. Thephotoacoustic pattern may include at least one photoacoustic signal. Themethod further includes analyzing the photoacoustic pattern to calculateat least one characteristic of the photoacoustic pattern, and comparingthe at least one characteristic of the photoacoustic pattern to a set ofcalibration patterns to determine one or more clot properties.

This method of detecting clots makes use of a negative photoacousticcontrast technique described herein. This technique exploits thesignificantly lower absorption of laser pulse energy by plateletscompared to red blood cells at selected ranges of pulse wavelengths. Itwas surprisingly discovered that circulating clots producedphotoacoustic signals that were significantly lower in magnitude thanthe background photoacoustic signals produced by the surroundingbloodstream. As a result, a sharp reduction in the magnitude ofphotoacoustic signals within a series of photoacoustic signals may beassociated with the detection of a clot in this technique.

In another aspect, a device for the continuous monitoring of acirculatory vessel of a living organism is provided that includes an invivo flow cytometer, a clot monitoring system, and an alarm system. Thein vivo flow cytometer includes a pulsed laser for pulsing a clot withinthe circulatory vessel and an ultrasound transducer for receiving aphotoacoustic signal emitted by a clot in response to the at least onepulse of laser energy. The clot may be pulsed at a pulse wavelengthranging between about 500 nm and about 600 nm. The clot monitoringsystem analyzes a photoacoustic pattern that includes at least onephotoacoustic signal and produces a detection signal if thephotoacoustic pattern indicates a clot. The alarm system provides analarm signal to the living organism in response to a detection signalproduced by the clot monitoring system.

In yet another aspect, a wearable device for the continuous monitoringof a circulatory vessel of a living organism is provided that includesan in vivo flow cytometer, a clot monitoring system, an alarm system,and a power source. The in vivo flow cytometer includes a pulsed laserfor pulsing a clot within the circulatory vessel and an ultrasoundtransducer for receiving a photoacoustic signal emitted by a clot inresponse to the at least one pulse of laser energy. The clot may bepulsed at a pulse wavelength ranging between about 500 nm and about 600nm. The clot monitoring system analyzes a photoacoustic pattern thatincludes at least one photoacoustic signal and produces a detectionsignal if the photoacoustic pattern indicates a clot. The alarm systemprovides an alarm signal to the living organism in response to adetection signal produced by the clot monitoring system. The powersource provides power to the in vivo flow cytometer, the clot monitoringsystem, and the alarm system. The device is secured to an appendage ofthe living organism chosen from an arm, a leg, a finger, a toe, a neck,and a head.

The devices and methods provided in various aspects of the presentinvention overcome limitations of previous devices and methods for clotdetection and treatment. Non-invasive photoacoustic flow cytometrydevices may be used in conjunction with the method of detecting a clotproperty to continuously monitor the blood flow through a circulatoryvessel of a living organism for extended periods. As a result, theentire blood volume may be assessed for clot properties such as thepresence, concentration, composition, or size of circulating clots asthe blood flow passes through a circulatory vessel. Further, theprogression of a clot-related health condition, or the efficacy of ananti-clot treatment may also be assessed over extended periods withoutneed for multiple invasive procedures such as repeated blood sampling.

Additional objectives, advantages and novel features will be set forthin the description which follows or will become apparent to thoseskilled in the art upon examination of the drawings and the detaileddescription which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures illustrate various aspects of the invention.

FIG. 1 is a flow chart of a photoacoustic in vivo flow cytometry method.

FIG. 2 is a schematic diagram illustrating an in vivo photoacoustic flowcytometry (PAFC) device.

FIGS. 3A-3D show the oscilloscope trace recordings of PA signals: (FIG.3A) from blood flow in a rat ear vessel with diameter of 50 μm, (FIG.3B) from skin surrounding a rat ear vessel before dye injection, (FIG.3C) from blood flow in a rat ear vessel 5 min after the injection ofLymphazurin, and (FIG. 3D) from the skin surrounding a rat ear vesselmeasured 20 min after dye injection.

FIG. 4 shows the PA signal detected from the monitoring of the bloodflow in a 50-μm rat ear microvessel after intravenous injection ofLymphazurin dye in the tail vein.

FIG. 5 shows the PA signal from circulating GNR in 50-μm rat mesenterymicrovessels as a function of post-injection time.

FIG. 6 is a graph of the normalized number of circulating GNR in bloodmicrovessels of the rat mesentery as a function of post-injection timeand a dashed curve showing averaged data (N=3).

FIG. 7 is a graph of the normalized number of circulating S. aureusbacteria in blood microvessels of the mouse ear as a function ofpost-injection time, for bacteria labeled using two different contrastsubstances, ICG dye and carbon nanotubes (CNTs).

FIG. 8 is a graph of the normalized number of circulating E. colibacteria in blood microvessels of the mouse ear as a function ofpost-injection time.

FIG. 9 shows the PA spectra of 50-μm mouse ear veins (empty circles),conventional absorption spectra of the B16F10 mouse melanoma cells withstrong pigmentation (upper dashed curve) and weak pigmentation (lowerdashed curve), spectra normalized using PA signals for the single mousemelanoma cells with strong pigmentation (black circles) and weakpigmentation (black squares), and absorption spectra for pure Hb andHbO₂ (fragments of solid curves in the spectral range 630-850 nm).

FIGS. 10A-10B are graphs showing the frequencies of circulating mousemelanoma cells (B16F10) detected with label-free PAFC in 50-μm mouse earveins, with a flow velocity of 5 mm/s, in mice with low (FIG. 10A) andhigh (FIG. 10B) melanin pigmentation as a function of post-injectiontime.

FIG. 11 is a summary of the PA signal rates from single melanoma cellsdetected in a mouse ear lymph microvessel 5 days after tumorinoculation.

FIG. 12 is a summary of the PA signal rates from a single RBC (whitebar) and lymphocytes (black bars) detected by PAFC in the lymph flow ofrat mesentery.

FIGS. 13A-13C show oscilloscope traces of the two-wavelength,time-resolved detection of PA signals from: (FIG. 13A) necroticlymphocytes labeled with gold nanorods absorbing 639 nm laser pulses,(FIG. 13B) apoptotic lymphocytes labeled with gold nanoshells absorbing865 nm laser pulses, and (FIG. 13C) live neutrophils labeled with carbonnanotubes absorbing both the 639 nm and the 865 nm laser pulses.

FIGS. 14A-14B show oscilloscope traces of the two-wavelength,time-resolved detection of PA signals from: (FIG. 14A) melanoma cellsabsorbing 865 nm and 639 nm laser pulses, and (FIG. 14B) red blood cellsabsorbing 865 nm and 639 nm laser pulses.

FIG. 15 is a summary of the PA signal rates from melanin particlesdetected in a mouse ear lymph microvessel 2 hours after injection.

FIG. 16 is a summary of the PA signal rates from single melanoma cellsdetected in a mouse ear lymph microvessel 4 weeks after tumorinoculation.

FIG. 17 is a summary of the PA signal amplitude generated by quantum dotmarkers as a function of laser fluence.

FIG. 18 is a summary of the PA signal amplitude generated by quantum dotmarkers as a function of the number of laser pulses.

FIG. 19 is an absorption spectrum of platelets compared to thecorresponding spectra of blood cells and gold nanotubes.

FIG. 20 is a schematic illustration of the subsystems of a device forthe continuous monitoring of a circulatory vessel of a living organism.

FIGS. 21A-21L include a series of optical images of slides containingsamples of red blood cells (FIG. 21A), non-aggregated platelets (FIG.21B), a platelet aggregate (FIG. 21C), and a platelet aggregate amongred blood cells (FIG. 21D). FIG. 21E, FIG. 21F, FIG. 21G, and FIG. 21Hare the photothermal signals obtained from the samples shown in FIGS.21A-21D, respectively. FIG. 21I, FIG. 21J, FIG. 21K, and FIG. 21L arethe photoacoustic signals obtained from the samples shown in FIGS.21A-21D, respectively.

FIG. 22 is a graph showing the spatial distribution of photoacousticsignals obtained from the average of eight scans of a collagen-inducedplatelet aggregate in a blood sample.

FIG. 23 is a graph of the negative contrast of collagen-induced plateletaggregates relative to surrounding blood cells in a blood sample as afunction of aggregate size.

FIGS. 24A-24B include an optical image (FIG. 24A) and a correspondingphotoacoustic scan (FIG. 24B) of a heterogeneous blood clot suspended ina whole blood sample.

FIGS. 25A-25B include optical images of a rat mesenteric vein beforeintravenous injection of collagen (FIG. 25A) and after the formation ofa collagen-induced clot (FIG. 25B).

FIGS. 26A-26D show oscilloscope traces of the photothermal signals (FIG.26A) and photoacoustic signals (FIG. 26B) measured from circulatingblood alone, and the photothermal signals (FIG. 26C) and photoacousticsignals (FIG. 26D) measured from circulating blood containing clots. Allsignals were generated after exposure to 605 nm laser pulses.

FIG. 27 is a graph of the photoacoustic signals generated using laserpulses of 532 nm on a blood vessel in a mouse ear, showing severalnegative signal dips corresponding to the detection of circulatingplatelet aggregates. The insets are enlarged views of single negativesignal dips.

FIGS. 28A-28C show oscilloscope traces of the photoacoustic signalsmeasured in a mouse carotid artery before the formation of clots (FIG.28A), after occlusion by a clot (FIG. 28B), and photoacoustic signalsfrom circulating heterogeneous clots in a mouse skin vessel (FIG. 28C).

FIG. 29 is a graph of the photoacoustic signals generated by laserpulses having wavelengths of 532 nm and 671 nm measured from bloodflowing through glass tubes that contained a mixture of target objects:circulating tumor cells (CTCs) labeled with gold nanorods, platelet-richclots, and labeled CTC-platelet clot complexes. An enlargement of thephotoacoustic signals associated with the detection of each type ofcirculating target object is shown in the inset graphs.

FIGS. 30A-30B include graph of the photoacoustic signals measured fromblood flow containing circulating clots in a mouse ear vessel before(FIG. 30A) and after (FIG. 30B) the application of a high-pass filter tothe photoacoustic signal data.

FIGS. 31A-31E are graphs of the photoacoustic signals measured fromblood flow containing circulating clots in a mouse ear vessel showingraw data (FIG. 31A), and after signal-averaging the signals from 16consecutive laser pulses (FIG. 31B), from 64 consecutive laser pulses(FIG. 31C), from 256 consecutive laser pulses (FIG. 31D), and from 1024consecutive laser pulses (FIG. 31E).

Corresponding reference characters indicate corresponding elements amongthe views of the drawings. The headings used in the figures should notbe interpreted to limit the scope of the claims.

DETAILED DESCRIPTION

The present invention relates to methods and devices for the continuousdetection of clots moving through a circulatory vessel of a livingorganism. The clots may be detected using devices and methods similar tothe photoacoustic flow cytometers (PAFC) described in U.S. patentapplication Ser. No. 12/334,217, which is hereby incorporated byreference in its entirety, and as described herein.

It has been discovered unexpectedly that circulating clots could bedetected using a negative photoacoustic contrast technique. In thistechnique, a circulating clot may be detected by pulsing the clot with alaser pulse having a wavelength that is absorbed by the platelets withinthe clot to a significantly lower degree relative to the absorption bysurrounding red blood cells. As a consequence, the photoacoustic signalproduced by a clot pulsed at this wavelength is significantly lower inmagnitude than the photoacoustic signals produced by surrounding redblood cells. By comparing the photoacoustic signals produced by theclots to the average background photoacoustic signal produced by redblood cells and surrounding tissues, the clots may be detected as anegative dip in the photoacoustic signal magnitude received by the PAFC.

The pattern of photoacoustic signals detected from circulating clots maybe analyzed to assess a clot property including, but not limited to, thesize or composition of a clot. For example, the magnitude or duration ofa negative dip in the photoacoustic signal may be analyzed to determinethe size of a clot. A characteristic signal pattern such as positivespike in the photoacoustic signal preceding and/or following a negativedip may provide information regarding the composition of a clot.

This method of detecting clots in the circulatory vessel of a livingorganism may be used to determine the presence and extent of circulatingclots in order to assess the organism's risk of adverse events such asstrokes or heart attacks, to assess the efficacy of an anti-clottreatment, or to direct the application of an anti-clot treatment. Forexample, the detection of a clot may trigger the pulsing of the detectedclot with a high-energy laser pulse in order to ablate the clot.

Further, a wearable device may be used to detect clots in thecirculatory vessel of the living organism. The device may include aminiaturized PAFC system that detects clots and issues an alarm in theevent of a detected clot. The alarm may provide a warning to theorganism and/or may summon medical attention. In another embodiment, thedevice may be used to continuously monitor the flow through a bloodvessel, and the detection of a clot may trigger a high-energy laserpulse at a wavelength and intensity sufficient to selectively eliminatethe detected clot.

Further descriptions of the methods and devices for the detection ofclots in the circulatory vessels of living organisms are providedherein.

I. Method of Detecting Clots

An in vivo device capable of detecting circulating clots in thecirculatory vessel of a living organism, such as a photoacoustic flowcytometer (PAFC) may be used in conjunction with a negativephotoacoustic contrast technique to detect the clots. A PAFC detectsphotoacoustic (PA) signals, typically in the form of ultrasound pulses,generated by circulating clots in response to the absorption of energyfrom a laser pulse directed at the clots. Subsequent analysis of thepattern of PA signals may be used to determine one or more clotproperties, including but not limited to the size and composition of theclot.

FIG. 1 is a flowchart summarizing a method of detecting a clot in acirculatory vessel of a living organism using a PAFC as the detectiondevice. A laser pulse is generated by the PAFC at step 201 and directedto the circulating clot at step 202. A PA signal is emitted by the clotin response to the absorption of the laser pulse. This PA signal isdetected by the PAFC using a sensor such as an ultrasound transducer atstep 203, and the detected PA signal may be optionally amplified and/ortransmitted to a recording device at step 205. The patterns of PAsignals are analyzed at step 206 to determine the presence orcomposition of the clots. For example, analysis of the PA signal patternmay identify a negative spike in the PA signal magnitude that mayindicate the detection of a circulating clot.

a. Photoacoustic Flow Cytometry

A photoacoustic flow cytometry (PAFC) device may be used to performnon-invasive in vivo flow cytometry in order to detect circulating clotswithin the vessel of a living organism. Referring to FIG. 2 , the PAFCdevice 100 is operated by illuminating a circulating clot 140 or othertarget object with one or more laser energy pulses 131, thereby inducingthe clot 140 to emit a photoacoustic (PA) signal 142. The PA signal 142typically falls within the ultrasound spectrum, with a range offrequencies between about 20 kHz and about 200 MHz. The PA signal 142emitted by the clots 140 may result from the absorption of the one ormore laser energy pulses 131 by a variety of mechanisms including, butnot limited to: single photon absorption, two photon absorption,multi-photon absorption, Coherent Anti-Stokes Raman Scattering (CARS),and combinations thereof.

The ultrasound transducer 150 detects the PA signal 142 emitted by theclot 140, and the output 160 from the ultrasound transducer 150 may beanalyzed using a data analysis system 158 residing on acomputer-readable media 156 to identify one or more clot propertiesincluding but not limited to the size of the clot 140 and thecomposition of the clot 140. In an aspect, an amplifier 152 may amplifythe output 160 of the ultrasound transducer 150 to produce an amplifiedsignal 164 and this amplified signal 164 may be analyzed using the dataanalysis system 158. In another aspect, the amplified signal 164 may bestored in a data recording system 154, as illustrated in FIG. 2 . In yetanother aspect, the data analysis system 158 may be used to retrieve andanalyze stored PA signal data 168.

Because the ultrasound waves of the PA signals travel freely throughmost biological tissues, the PAFC device 100 may be used to detectcirculating clots 140 in circulatory vessels as deep as 15 cm below theexternal surface of the organism. Further, because the laser power usedby the PAFC device 100 is relatively low, the PAFC device 100 may beoperated for extended time periods with minimal damage to circulatingcells and surrounding tissues. A PAFC device 100 using a negativephotoacoustic contrast technique may be used for the continuousmonitoring of clots for the early diagnosis and treatment of strokes,heart attacks, and other clot-related disorders.

b. Negative Photoacoustic Contrast Technique of Clot Detection

In an aspect, a negative photoacoustic contrast technique may be used todetect clots in a circulatory vessel of a living organism and todetermine one or more clot properties based on an analysis ofphotoacoustic patterns associated with the clots. As described above,the in vivo photoacoustic flow cytometry (PAFC) device may be used tocollect laser-excited photoacoustic (PA) signals emitted by at least onetype of target object, which may be a clot, within a circulatory vessel.The photoacoustic patterns comprising PA signals collected over one ormore consecutive laser pulses may be analyzed to determine at least oneclot property. Non-limiting examples of clot properties that may bedetermined using the analysis of a photoacoustic pattern associated withthe clot include: the size of the clot, the type or composition of theclot, the quantity of clots, the concentration of clots, the flow speedof the clots within the circulatory vessel, the age of the clot, theorigin of the clot, and combinations thereof.

It was unexpectedly discovered that platelets, which typically make up asignificant portion of clots, emit a relatively weak PA signal comparedto the stronger background PA signals emitted by other target objectssuch as red blood cells and other blood components in response to laserpulses at the same wavelength. For example, the photoacoustic patternemitted by a clot suspended within a blood sample is shown in FIG. 22 .The dashed line in this figure represents the average PA signal strengthemitted by the surrounding blood sample, which predominantly includes PAsignals emitted by red blood cells. However, the PA signals emitted bythe clot are significantly lower in strength than the background bloodPA signal strength. This negative spike in PA signal strength indicatesthe detection of a clot. “Negative spike”, as used herein, refers to anabrupt reduction in PA signal strength within a photoacoustic pattern.For example, the photoacoustic pattern illustrated in FIG. 22 includes anegative spike 2202. The analysis of photoacoustic patterns associatedwith the clot, including but not limited to the shape of the negative PAsignal spike, the duration of the negative PA signal spike, and themagnitude of the negative PA signal spike, may be used to identifyvarious clot properties.

i. Photoacoustic Patterns

Photoacoustic (PA) patterns may be analyzed to determine a clot propertysuch as clot size or composition. A PA pattern, as used herein, refersto a time series of one or more PA signals emitted by one or more targetobjects in response to the absorption of one or more consecutive laserpulses delivered at one or more pulse wavelengths. In general, PApatterns may be associated with a particular type of target objectincluding, but not limited to, clots. Different types of target objectstypically possess unique combinations of pigments and sub-cellularstructures that absorb laser pulses and emit PA signals differently.Each type of target object may be uniquely identified by its distinctivePA pattern, as well the differences in PA patterns elicited by differentwavelengths of laser pulses by the same target object.

The contrast of the PA patterns of the clots relative to surroundingcells and tissues typically result from PA signal amplitudes from theclots that are significantly lower than the PA signal amplitudes fromsurrounding cells and tissues. The PA contrast of a clot, as definedherein, refers to the difference between the PA signal amplitude from aclot and the PA signal amplitude from surrounding blood cells andtissues. Circulating clots may be detected through the time-resolvedmonitoring of dynamic decreases of the PA signal amplitude, due to theattenuated PA signal amplitude emitted by clots relative to the PAsignal amplitude emitted by surrounding red blood cells. The decreasedPA signal amplitude emitted by clots is due to the lower energyabsorption of platelets (a component of blood clots) relative to theenergy absorption of red blood cells (a common cell type in typicalblood flows) for laser pulses in the visible and near-infrared spectralrange, as discussed above.

Non-limiting examples of PA patterns suitable for identifying a clotproperty include pattern shape, frequency spectrum of the pattern, PAsignal amplitude, as well as phase shift and/or time delay between oneor more laser pulses and the received PA signal within a pattern. The PAsignals within a PA pattern may be discriminated between PA signalsdetected from clots and background PA signals detected from surroundingcells and circulatory vessel walls and tissues. In an aspect, the PApattern is the amplitude of the negative spike associated with a clot.In this aspect, the PA signal amplitude detected from the clot may beless than about 50% of the signal amplitude detected from thesurrounding target objects and tissues. In another aspect, the PA signalamplitude detected from the clot is less than about 10% of the signalamplitude detected from the surrounding target objects and tissues. Inyet another aspect, the PA signal amplitude detected from the clot isless than about 1% of the signal amplitude detected from the surroundingtarget objects and tissues. Photoabsorbant marker particles may beattached to the clots to enhance the degree of contrast of the PAsignals of the clots relative to the PA signals of the surroundingtarget objects such as blood and tissues.

In another aspect, the contrast of the PA signal of a clot may be usedto determine the size of a clot. For example, FIG. 23 is a summary ofthe negative contrast of a series of clots of different sizes. Based onthese data, larger clots were associated with a higher degree ofnegative contrast. Other indications of the overall size of a clot mayinclude, but are not limited to, the absolute magnitude of the PA signalof a clot and the duration of a negative PA spike.

In yet another aspect, the shape of a PA pattern may be used to indicatethe composition of a clot. The composition of a clot may depend on oneor more factors including but not limited to the age of the clot, thesize of the clot, and the presence or absence of surrounding cells suchas red blood cells, white blood cells, and circulating tumor cells,which may be incorporated into the clot. The composition of a clot mayinclude the proportion of platelets relative to other clot components,the spatial distribution of these other components within the clot, andany combination thereof. For example, FIG. 24 is a PA pattern resultingfrom a photoacoustic scan of a clot within a blood sample. The PApattern in FIG. 24 is characterized by a positive PA spike followed by anegative PA signal dip followed by a second positive PA spike. In thisfigure, the positive PA spikes indicate a high concentration of redblood cells within the peripheral surface of the clot that emits strongPA signals. The negative PA signal dip indicates a platelet-rich centerregion of the clot. By contrast, the negative PA dips shown in FIG. 27lack the prominent positive PA spikes preceding and following thenegative PA dip, indicating that the detected clots in FIG. 27 arerelatively rich in platelets.

In another aspect, two or more photoacoustic patterns detected inresponse to laser pulses from two or more pulse wavelengths may becompared to determine the composition of a clot or other clot property.For example, FIG. 29 is a summary of the PA signals received in responseto laser pulses of two different pulse wavelengths: 532 nm and 671 nmpulses. As illustrated in FIG. 29 , red blood cells are responsive to532 nm pulses, circulating tumor cells (CTCs) are responsive to 671 nmpulses, and platelets are relatively unresponsive to either pulsewavelength, resulting in negative spikes in PA patterns associated withplatelets. By comparing the PA patterns resulting from different pulsewavelengths, it may be determined whether a clot contains mostlyplatelets, as indicated by the photoacoustic patterns in the left insetgraph, or whether a clot further includes CTCs, as indicated by thephotoacoustic patterns in the center inset graph of FIG. 29 .

The velocity of the flow within a circulation vessel may be determinedusing PA patterns including but not limited to the PA signal duration,the PA frequency shift, or the time delay between two PA signalsproduced by a single target object pulsed by two distinct laser pulsesapplied at a known separation distance. The speed of movement of a clotwithin the circulation vessel may be determined by identifying thepresence of a clot using the analysis of PA patterns as described above,and then determining the velocity of the identified clot using PApatterns including but not limited to the PA signal duration, the PAfrequency shift, or the time delay between two PA signals produced by asingle clot pulsed by two distinct laser pulses applied at a knownseparation distance.

In an aspect, the overall flow velocity and the speed of movement of aclot within a circulatory vessel may be determined and compared toassess clot properties. For example, a speed of movement of a clot thatis comparable to the overall flow speed within the circulatory vesselmay indicate a small, freely-moving clot, whereas a clot speed that ismuch slower than the surrounding flow speed may indicate a larger clotor a rolling clot. Other factors may be considered in conjunction withthe clot speed to determine additional clot properties such as the ageand origin of the clot. For example, if the organism has other factorssuch as a previous history of clots or known adhered clots elsewhere inthe organism, the detection of small, fast moving clots may indicatethat these fast-moving clots may have fractured from a larger,slower-moving clot or an adhered clot elsewhere within the organism.

The photoacoustic patterns may be analyzed by comparing thephotoacoustic patterns to a set of calibration patterns obtained fromclots having known clot characteristics including, but not limited to,clot size and clot composition. The set of calibration patterns mayinclude patterns obtained at various PAFC conditions including, but notlimited to, laser pulse wavelength, laser pulse duration, laser pulsefluence, frequency of laser pulses, and any combination thereof.

ii. Selection of Pulse Wavelength

A critical factor in the negative photoacoustic contrast technique isthe selection of a laser pulse wavelength that results in PA signalsemitted by clots that are significantly lower than the PA signalsemitted by other blood components including but not limited to red bloodcells and other target objects within the circulatory vessel of theliving organism. In an aspect, the laser wavelength may be selected forthe negative photoacoustic contrast technique based on the difference inabsorption of the clots relative to other target objects within thecirculatory vessel.

Clots typically include platelets as part of their composition alongwith other blood components including, but not limited to, red bloodcells and circulating tumor cells. In an aspect, a laser wavelength thatis not well absorbed by platelets, but is strongly absorbed by otherblood components may be selected as the pulse wavelength for thedetection of clots using a negative photoacoustic contrast technique.Without being limited to any particular theory, the higher absorbance ofa laser pulse by a target object typically results in the emission of astronger PA signal by that target object. Referring to FIG. 19 , theabsorption of laser energy for all wavelengths from about 400 nm toabout 1000 nm by blood is significantly higher than the absorption byplatelets. In particular, blood absorbs at least 10-100 times more laserenergy at wavelengths ranging from about 500 nm to about 600 nm thanplatelets. In an aspect, the pulse wavelength used to detect a clotproperty may fall within a strong blood absorption band and may rangebetween about 500 nm and about 600 nm or between about 530 nm and about580 nm. In another aspect, the pulse wavelength may be selected toinduce a photoacoustic signal from the clot that is at least 5 timeslower in magnitude than the photoacoustic signal obtained fromsurrounding red blood cells induced by the pulse wavelength. In anadditional aspect, the pulse wavelength used to detect a clot propertyin a circulatory vessel may be about 532 nm, the wavelength resulting inthe maximum contrast between blood and platelets according to the datasummarized in FIG. 19 .

In yet another aspect, PA signals induced by two or more laser pulses inwhich each laser pulse has a unique pulse wavelength that differs fromthe pulse wavelength of the other pulses may be used to identify a clotproperty. In this aspect, at least one of the unique pulse wavelengthsmay be strongly absorbed by a non-platelet target object type, includingbut not limited to circulating tumor cells. PA patterns elicited by thetwo or more pulse wavelengths may be compared to detect a clot propertyas discussed herein above.

II. Clots and Other Target Objects

In various aspects, the negative photoacoustic contrast technique may beused to detect clots within the circulatory vessels of living organisms.The clots may be detected at a resolution of at least about 1 clot permL of blood. Using this method to continuously monitor the blood as itflows through a circulatory vessel may detect clots at a concentrationof about 5 clots or less within the entire blood volume of the organism.The individual clots detected using this method may be as small as about20 μm.

In an aspect, the clots may be detected within circulatory vessels at adepth ranging from about 10 μm to about 15 cm below the surface of theskin. Non-limiting examples of circulatory vessels include capillaries,arterioles, venules, arteries, veins, and lymphatic vessels. Thediameters of the circulatory vessels may range between about 10 μm andabout 2 cm. The diameter of the circulatory vessel may be selected inorder to enhance the negative contrast of the clots relative to thesurrounding blood flow. Leukocytes and the plasma layer within the bloodflow may also produce significantly lower photoacoustic signals comparedto surrounding red blood cells, resulting in negative contrast signalsthat confound the analysis techniques used to detect clots. Within smallcirculatory vessels such as capillaries, the confounding negativecontrast from leukocytes and plasma is more pronounced; this confoundingnegative contrast is attenuated in larger-diameter circulatory vessels.In an aspect, the circulatory vessels in which clots are detected usingthe negative photoacoustic contrast technique may have a mean diameterof at least about 25 μm.

The circulatory vessels may be located in various organs and tissues,including, but not limited to skin, lips, eyelid, interdigital membrane,retina, ear, nail pad, scrotum, brain, breast, prostate, lung, colon,spleen, liver, kidney, pancreas, heart, testicles, ovaries, lungs,uterus, skeletal muscle, smooth muscle, and bladder. Clot properties maybe detected in the circulatory vessels of any organism that possessescells circulating in vessels or sinuses chosen from the group oforganisms including mammals, reptiles, birds, amphibians, fish,mollusks, insects, arachnids, annelids, arthropods, roundworms, andflatworms.

The clots and other target objects detected in various aspects mayinclude but are not limited to unlabeled biological cells, biologicalcell products, unbound contrast agents, biological cells labeled usingcontrast agents, aggregations of cells, platelet-rich white clots, redblood cell-rich clots, heterogeneous clots comprising platelets and oneor more other target object types, and any combination thereof. Thetarget objects may be unlabeled endogenous or exogenous biological cellsor cell products including but not limited to normal, apoptotic andnecrotic red blood cells and white blood cells; aggregated red bloodcells or clots; infected cells; inflamed cells; stem cells; dendriticcells; platelets; metastatic cancer cells resulting from melanoma,leukemia, breast cancer, prostate cancer, ovarian cancer, and testicularcancer; bacteria; viruses; fungal cells; protozoa; microorganisms;pathogens; animal cells; plant cells; and leukocytes activated byvarious antigens during an inflammatory reaction and combinationsthereof.

The target objects may also be biological cell products, including butnot limited to products resulting from cell metabolism or apoptosis,cytokines or chemokines associated with the response of immune systemcells to infection, exotoxins and endotoxins produced during infections,specific gene markers of cells such as tyrosinase mRNA and p97associated with cancer cells, MelanA/Mart1 produced by melanoma cells,PSA produced by prostate cancer, and cytokeratins produced by breastcarcinoma.

The target objects may also be contrast agents chosen from the groupincluding indocyanine green dye, melanin, fluoroscein isothiocyanate(FITC) dye, Evans blue dye, Lymphazurin dye, trypan blue dye, methyleneblue dye, propidium iodide, Annexin, Oregon Green, C3, Cy5, Cy7, NeutralRed dye, phenol red dye, AlexaFluor dye, Texas red dye, goldnanospheres, gold nanoshells, gold nanorods, gold cages, carbonnanoparticles, prefluorocarbon nanoparticles, carbon nanotubes, carbonnanohorns, magnetic nanoparticles, quantum dots, binary gold-carbonnanotube nanoparticles, multilayer nanoparticles, clusterednanoparticles, liposomes, liposomes loaded with contrast dyes, liposomesloaded with nanoparticles, micelles, micelles loaded with contrast dyes,micelles loaded with nanoparticles, microbubbles, microbubbles loadedwith contrast dyes, microbubbles loaded with nanoparticles, dendrimers,aquasomes, lipopolyplexes, nanoemulsions, polymeric nanoparticles, andcombinations thereof.

The target objects may also be labeled cells, clots, platelets, or othertarget objects listed herein above, marked with molecular markers andtags comprised of contrast agents listed herein above. The molecularmarkers or tags may be attached to the cells without modification, orthe contrast agents may be functionalized for binding to the cells usingmolecules including, but not limited to: antibodies, proteins, folates,ligands for specific cell receptors, receptors, peptides, viramines,wheat germ agglutinin, and combinations thereof. Non-limiting examplesof suitable ligands include: ligands specific to folate, epithelial celladhesion molecule (Ep-CAM), Hep-2, PAR, CD44, epidermal growth factorreceptor (EGFR), as well as receptors of cancer cells, stem cellsreceptors, protein A receptors of Staphylococcus aureus, chitinreceptors of yeasts, ligands specific to blood or lymphatic cellendothelial markers, as well as polysaccharide and siderophore receptorsof bacteria.

Exogenous target objects such as unbound contrast agents and exogenousunlabeled biological cells may be introduced into the circulatoryvessels of the organism parenterally, orally, intradermally,subcutaneously, or by intravenous or intraperitoneal administration.

III. Photoacoustic Flow Cytometry Device

A photoacoustic flow cytometry (PAFC) device may be used to detect theclots within the circulatory vessel of a living organism using thenegative photoacoustic contrast technique described herein.

a. Overview of PAFC Device

FIG. 2 is a schematic illustration of a PAFC device 100 used in anaspect to detect clots within a circulatory vessel. The PAFC device 100may include a tunable wavelength pulsed laser 120 capable of emittinglight energy 126 ranging between wavelengths of about 400 nm and about2500 nm. The tunable wavelength pulsed laser source 120 includes apulsed laser 122, and may further include an optical module 124 toconvert the wavelength, pulse rate, or both wavelength and pulse rate ofthe laser pulse 123 emitted by the pulsed laser 122 to desired values.In addition, the PAFC device 100 includes optical elements 130 such aslenses or optic fibers to direct the laser light 131 to the clots 140within the area of interest 132. The PAFC device 100 also includes atleast one ultrasound transducer 150 to detect photoacoustic signals 142emitted by the clots 140. A magnet 110 may also be included in order tolocally enrich the concentration of clots or other target objects withinthe area of interest 132 detected by the PAFC device 100. The PAFCdevice 100 may optionally include an amplifier 152, a data recordingsystem 154, and computer readable media 156 with stored data analysissoftware 158.

The PAFC device 100 may further include additional elements including,but not limited to photodetectors, additional lasers and optics, andadditional analysis software associated with other in vivo flowcytometry methods that detect the clots or other target objects usingalternative detection methods. Non-limiting examples of alternativedetection methods include methods that make use of the conventional andRaman scattering of the laser pulses by the clots or other targetobjects, photothermal effects induced by laser pulses on the clots orother target objects, and the fluorescence of the clots or other targetobjects induced by absorbed laser pulses. In an aspect, the PAFC device100 may be configured to simultaneously detect cells using photoacousticmethods, photothermal methods, light scattered by target objects,induced fluorescence of target objects, and any combination thereof.

b. Data Processing

Data analysis software 158 may process data stored on the data recordingsystem 154, the signal output from the ultrasonic transducer 150, theamplified signal output from the amplifier 152, or combinations thereof.The data analysis software 158 may also function as an amplifier, a datastorage device, and combinations thereof. Any data analysis softwarecapable of processing data obtained at signal acquisition frequenciesranging between about 20 Hz and 200 MHz may be used, including but notlimited to user-written software and commercially available analysissoftware. Non-limiting examples of commercially available analysissoftware include Matlab (The MathWorks, Inc., USA), Mathematica (WolframResearch, Inc., USA), Labview (National Instrument, USA), Avisoft(Avisoft Bioacoustics, Germany), and TomoView (Olympus NDT Inc., USA).

Any known method of processing the fluctuating PA signal data may beused in various aspects. For example, if a relatively high laser pulserate is used to generate the PA signals using the PAFC device 100, dataprocessing methods including signal filtering and signal averaging maybe used to eliminate signal fluctuations or signal noise resulting fromconfounding factors including but not limited to movements of the livingorganism due to breathing, blood pulse, or other movements, as well asvariation due to multiple PA signals received from the same clot orother target objects.

In one aspect, a series of multiple PA signals may be signal averaged toreduce fluctuations in the data series. Signal averaging is definedherein as the replacement of each PA signal value at a particular timewith an average of a group of two or more PA signals, in which theaveraged group of signals includes the original signal as well as one ormore PA signals immediately preceding and/or following the original PAsignal. The number of adjacent signals used in the signal averagingprocess may depend on at least one factor including, but not limited to:the frequency of fluctuations within the signal data, the number ofsignals per second acquired by the data analysis software, the laserpulse rate used to generate the PA signals, and any combination thereof.In this aspect, the number of adjacent PA signals that may be averagedin the signal averaging process may range from 2 PA signals to about2,500 PA signals. If the laser pulse rate used by the PAFC is in excessof 10 KHz, even larger groups of PA signals may be averaged.

In another aspect, the PA signal data may be subjected to signalfiltration using any known signal filter, including but not limited to ahigh-pass filter, a low-pass filter, and combinations thereof. Ahigh-pass filter is defined herein as a signal processing technique thateliminates PA signal data fluctuating below a specified cutofffrequency. A low-pass filter is defined herein as a signal processingtechnique that eliminates PA signal data fluctuating above a specifiedcutoff frequency. The cutoff frequency may be specified by the user, orthe cutoff frequency may be dynamically determined by the data analysissoftware. For example, the PA signal data may be subjected to ahigh-pass filter with a threshold frequency of about 100 Hz to eliminatelow-frequency data fluctuations due to confounding factors such asmovements of the organism due to breathing and blood pulse.

IV. Method for Eliminating Circulating Clots In Vivo

In an aspect, a method for the elimination of circulating clots in acirculation vessel of a living organism is provided that includesdetecting the clots in the circulation vessel, and then pulsing thedetected clot with at least one high-intensity pulse of laser energy toeliminate the detected clot. The high-intensity pulse may be deliveredat significantly higher laser fluence than the laser fluence used forthe detection of the clot, and may be delivered at a wavelength at whichthe clot has higher absorbance efficiency. In this method, the detectionof a clot triggers a pulse of laser energy that is delivered to thedetected clot at a wavelength and fluence sufficient to cause thedestruction of the detected clot. The method may further includemonitoring the frequency of detection of clots circulating through thecirculatory vessels, and terminating the method when the frequency ofdetection of the clots falls below a threshold level. In an aspect, thismethod of eliminating clots may be terminated when the frequency ofdetection of clots falls below a rate ranging between about 10⁻³clots/min and about 10² clots/min.

V. Device for Continuous Monitoring of a Circulatory Vessel

In an aspect, a device for the continuous monitoring of a circulatoryvessel of a living organism is provided. This device 300, shown as ablock diagram in FIG. 20 , includes an in vivo flow cytometer 302, aclot monitoring system 304, and an alarm system 306. This device 300monitors the blood flow through a circulatory vessel and detects clotsusing the negative photoacoustic contrast techniques describedpreviously herein. Upon the detection of a clot in the circulatoryvessel, the alarm system 306 of the device 302 issues an alarm signal tonotify the wearer or operator of the device 300, or to notify medicalpersonnel.

The in vivo flow cytometer 302 is similar to the PAFC devices describedpreviously herein. In this aspect, the in vivo flow cytometer 302includes a pulsed laser for pulsing the clot within the circulatoryvessel. In order to enhance the effectiveness of the negativephotoacoustic contrast technique of clot detection, the pulsed laser maydeliver at least one pulse of laser energy at a pulse wavelength rangingfrom about 500 nm to about 600 nm, as described herein. The in vivo flowcytometer 302 further includes an ultrasound transducer similar to theultrasonic transducers described herein and as described in U.S. patentapplication Ser. No. 12/945,576. The ultrasonic transducer receives aphotoacoustic (PA) signal emitted by the clot in response to the atleast one pulse of laser energy.

The clot monitoring system 304 processes the PA signals detected by invivo flow cytometer 302 to determine whether the pattern of PA signalsindicates the detection of a clot. The photoacoustic pattern comprisingat least one photoacoustic signal is analyzed using processes andmethods similar to those described herein above. If a clot is detectedby the clot monitoring system 304, a detection signal is produced.

The alarm system 306 receives detection signals from the clot monitoringsystem 304. Upon receiving a detection signal, the alarm system 306 mayissue an alarm signal to the living organism. The alarm signal may beissued in response to a single detection signal, or after processing thedetection signal using known methods. For example, the alarm signal maybe issued after receiving a detection signal of sufficient magnitude toindicate an alarm condition, or after receiving two or more detectionsignals at a sufficiently high frequency.

The alarm signal may comprise an indication to the device's wearer ofthe detection of a clot and may be continuous or intermittent in nature.Any known indication may be used, including but not limited to a visualdisplay, an audible sound, a vibration, a signal to summon medicalassistance, and any combination thereof. In an aspect, the device 300may further include a communication link to a hospital, medical center,or any other known location of medical assistance personnel in order totransmit the alarm signal to summon medical assistance. Any knowncommunication link may be used to transmit the alarm signal, includingbut not limited to wireless communication technologies such as Bluetoothsignals, cellular signals, wireless Internet signals, and anycombination thereof.

In another aspect, the device 300 may further comprise a clot treatmentsystem 308 for initiating a clot treatment in response to a detectionsignal and/or alarm signal. In this aspect, the clot treatment system308 may receive a detection signal from the clot monitoring system 304and/or an alarm signal from the alarm system 306, as illustrated in FIG.20 , and initiate a clot treatment in response. The clot treatment maybe any known clot treatment including, but not limited to, theadministration of an anti-clotting medication, or the pulsing of theclot with a high-intensity laser pulse at a laser fluence sufficient toeliminate the clot, as described previously herein.

The anti-clotting medication may be administered upon the receipt of asingle signal, or the clot treatment system 308 may locally process thealarm and/or detection signals and administer anti-clotting medicationbased on a processed signal quantity including but not limited to thesignal magnitude, the frequency of signals, the elapsed time since theprevious signal, and any combination thereof. The anti-clottingmedication may be administered in any known manner, including but notlimited to the administration of discrete dosages, continuousadministration, and any combination thereof. The discrete dosage amountand continuous rate of administration may be constant, may vary inaccordance with a predetermined schedule, may be specified by the weareror operator of the device 300, may be dynamically controlled by devicefeedback such as the frequency of alarm or detection signals, or anycombination thereof.

The device 300 may be a laboratory-based or hospital-based device, orthe device 300 may be a portable and self-contained device. In oneaspect, the device 300 may be a self-contained wearable device to beworn by a living organism to continuously monitor a circulatory vesselfor clots. In this aspect the self-contained wearable device may furtherinclude a power source including, but not limited to a battery. Thedevice 300 may be removably attached to a fixed location on the livingorganism using known methods including but not limited to straps,adhesive patches and adhesive strips. The device 300 may be removablyattached at any external location on a living organism that is situatedwithin a suitable distance of a circulatory vessel for continuousmonitoring using the methods described previously herein. Non-limitingexamples of suitable external locations include the neck, wrist,forearm, upper arm, hand, foot, ankle, lip, ear, chest, abdomen, eye,scalp, and leg.

EXAMPLES

The following examples illustrate aspects of the invention.

Example 1. Determination of Laser-Induced Cell Damage to Blood Cells andSubsequent Cell Viability Associated with In Vitro Photothermal (PT)Imaging

To determine whether the laser pulses associated with in vivo flowcytometry caused any significant damage to cells or tissues of theorganism, the following experiment was conducted. The laser-induceddamage threshold of single cells was evaluated as a function of thepumped-laser energy levels at a range of wavelengths using establishedmethods (Zharov and Lapotko 2005, Lapotko and Zharov 2005). In vitromeasurements of specific changes in photothermal (PT) images and PTresponses from individual cells were used to determine cell damage.During the PT imaging, individual cells were illuminated with a pulse oflaser light at a specified energy level and wavelength. After absorbingthe energy of the laser pulse, the short-term temperature of the cellincreased by as much as 5° C. The laser-induced temperature-dependentrefractive heterogeneity in the vicinity of cells caused defocusing of acollinear He—Ne laser probe beam (model 117A; Spectra-Physics, Inc.; 633nm, 1.4 mW) that illuminated the cell immediately after the initiallaser pulse. This defocusing caused a subsequent reduction in the beam'sintensity at its center, which was detected with a photodiode (C5658;Hamamatsu Corp.) through a 0.5-mm-diameter pinhole.

PT measurements were performed in vitro using mouse blood cells insuspension on conventional microscope slides. To simulate blood flowconditions, a flow module fitted with a syringe pump—driven system (KDScientific, Inc.) was used with glass microtubes of different diametersin the range of 30 μm to 4 mm that provided flow velocities of 1-10cm/sec, which were representative of the diameters and flow rates ofanimal microvessels.

Individual cells flowing through the glass microtubes were exposed to an8 ns burst of laser light in a 20-μm circular or elongated beam at avariety of wavelengths ranging between 420 nm and 2300 nm. At eachwavelength of the initial laser pulse, the laser fluence, defined as theenergy contained in the laser beam, was varied between 0.1 mJ/cm² and1000 J/cm². Damage to the cells was determined by assessing the changesin the PT imaging response of cells to laser pulses of increasingfluence. In addition, cell viability after exposure to laser energy wasassessed using a conventional trypan blue exclusion assay. Cellulardamage was quantified as ED50, the level of laser fluence at which 50%of the measured cells sustained photodamage in vitro. The ED50 valuesmeasured for rat red blood cells (RBC), white blood cells (WBC) and K562blast cells using laser pulses in the visible light spectrum aresummarized in Table 1. The ED50 values measured for rat red blood cells(RBC) and white blood cells (WBC) using laser pulses in thenear-infrared (NIR) light spectrum are summarized in Table 2.

TABLE 1 Photodamage thresholds for single rat blood cells in the visiblelight spectrum. Wavelength of Photodamage threshold ED50 (J/cm²) laserpulse (nm) Rat RBC Rat WBC Rat K562 blast cell 417 1.5 12 36 555 5 42 90

TABLE 2 Photodamage thresholds for single rat blood cells in near-IRspectral range. Wavelength of Photodamage threshold ED50 (J/cm²) laserpulse (nm) Rat RBCs Rat WBCs 740 6.9 21.7 760 6.8 — 780 17.7 152 80017.5 219 820 28.0 251 840 43.5 860 43.8 730 880 76.5 — 900 69.4 — 92077.7 357 960 33.5 48.8

In the visible spectral range, the relatively strong light-absorbingRBCs sustained cell damage at much lower intensities of laser energy,resulting in ED50 values that were about an order of magnitude lowerthan the ED50 values measured for WBC or K562 blast cells. In the NIRspectral range (wavelengths above about 800 nm), most cells includingRBC have minimal absorption. As a result, neither the RBCs nor WBCssustained damage in the NIR range until much higher laser energy levelscompared to the energy levels at which cellular damage occurred to cellsexposed to laser energy in the visible spectrum. The damage thresholds(ED50) for RBCs and WBCs in the spectral range of 860-920 nm were morethan one order magnitude higher compared to those in the visiblespectrum as shown in Tables 1 and 2.

The results of this experiment established the levels of laser energy atwhich laser-induced cellular damage may occur. In the NIR spectrum, inwhich cells exhibited the strongest photoacoustic effects, the damagethresholds are several orders of magnitude above the maximum safetylevel of approximately 20-100 mJ/cm² set by ANSI safety standards. Thus,photoacoustic flow cytometry may be performed in vivo with little riskof cell or tissue damage.

Example 2. Detection of Contrast Dye Circulating in Mice by Prototype InVivo Photoacoustic Flow Cytometry System

The following experiment was conducted to demonstrate the feasibility ofin vivo photoacoustic flow cytometry (PAFC) for real-time, quantitativemonitoring in the blood circulation of a conventional contrast agent,Lymphazurin. In this experiment, a prototype PAFC system was used todetect Lymphazurin circulating in the blood vessels of a mouse ear.

The prototype PAFC system was built on the platform of an Olympus BX51microscope (Olympus America, Inc.) and a tunable optical parametricoscillator (OPO) pumped by a Nd:YAG laser (Lotis Ltd., Minsk, Belarus).The general layout of the PAFC system is shown schematically in FIG. 2 .Laser pulses were produced at an 8 ns pulse width, a regular repetitionrate of 10 Hz with the ability to provide short-term pulses at 50 Hz,and a wavelength in the range of 420-2,300 nm. Laser energy was directedto the blood vessels using a conventional lens and/or an optical fiber.PA signals emitted by the cells were detected by ultrasound transducers(unfocused Panametrics model XMS-310, 10 MHz; focused cylindricalPanametrics model V312-SM, 10 MHz, focused lengths of 6 mm, 12 mm, and55 mm; and customized resonance transducers), and the ultrasoundtransducer outputs were conditioned by an amplifier (Panametrics model5662, bandwidth 50 kHz-5 MHz; Panametrics model 5678, bandwidth 50kHz-40 MHz; customized amplifiers with adjustable high and low frequencyboundaries in the range to 50-200 KHz and 1-20 MHz, respectively;resonance bandwidth of 0.3-1.0 MHz). The amplifier output signals wererecorded with a Boxcar data acquisition system (Stanford ResearchSystems, Inc.) and a Tektronix TDS 3032B oscilloscope, and were analyzedusing standard and customized software. The Boxcar data acquisitiontechnique provided averaging of the PA pulses emitted by cells in theblood vessels, and discriminated the PA waves from background signalsoriginating from surrounding tissue on the basis of the difference intime delays between the two signals. The signals from the oscilloscopescreen were recorded with a digital camera (Sony, Inc.) and video camera(JVC, Inc.).

A high-speed computer (Dell Precision 690 workstation with a quadcoreprocessor, 4 GB of RAM, and a Windows Vista 64 bit operating system) anddigitizer (National Instruments PCI-5124 high speed digitizer) were usedto acquire the PA signal data from the PAFC device. National Instrumentssoftware (Labview Version 8.5 and NI Scope Version 3.4) was used tocontrol the digitizer and to create a data logging user interface. Thehardware and supporting program were capable of collecting data at arate of 200 megasamples per second, corresponding to a time resolutionof 5 ns.

A laser beam with a circular cross section and a diameter ofapproximately 50 μm, a wavelength of 650 nm, and a fluence of 35 mJ/cm²was used to illuminate the flow in the blood vessels. The 650 nmwavelength used was near the wavelength of maximum absorption ofLymphazurin, the contrast dye used in this experiment, and waswell-separated from the wavelengths of maximum absorption of other bloodcomponents. Navigation of the laser beams was controlled withtransmission digital microscopy (TDM) at a resolution of approximately300 nm using a Cascade 650 CCD camera (Photometrics).

The in vivo experiments described below were performed using a nudemouse ear model. PAFC detection was performed using relativelytransparent, 270 μm thick mouse ears with well-distinguished bloodmicrovessels. The ear blood microvessels examined were located at adepth of 30-100 μm, had diameters in the range of 30-50 μm, and bloodvelocities of 1-5 mm/sec. After undergoing anesthesia usingketamine/xylazine at a dosage of 50/10 mg/kg, each mouse was placed on acustomized heated microscope stage, together with a topical applicationof warm water, which provided acoustic matching between the transducerand mouse ear.

After anaesthetizing each mouse and placing the mouse on the microscopestage as described above, 200 μL of a 1% aqueous solution of Lymphazurincontrast agent (Ben Venue Labs Inc., USA) was injected into the tailvein of the mouse. PAFC measurements of the circulating dye wereperformed at a laser pulse wavelength of 650 nm. FIG. 3 showsoscilloscope traces of PAFC signals from the blood vessels andsurrounding tissues in the rat ear before and after injection withLymphazurin. Prior to injection, the maximum 240 mV PA signals fromblood vessels, shown in FIG. 3A, were approximately 1.5 times higherthan the 160 mV PA background signals from surrounding tissue, shown inFIG. 3B. Maximum PA signals from the blood vessel after dyeadministration, shown in FIG. 3C, increased approximately three-foldover pre-injection levels. The PA signals from tissue around vesselsafter dye injections, shown in FIG. 3D, gradually increasedapproximately 2.5-fold over pre-injection levels during the first 15-20minutes, and then remained relatively constant for the next 1-1.5 hours,probably due to the passage of the Lymphazurin out of the blood vesselsand into nearby lymphatic vessels.

FIG. 4 summarizes the maximum PAFC signals from Lymphazurin compared tobackground PAFC signals from untreated blood vessels, observed for onehour after the injection of Lymphazurin. As shown in FIG. 4 , continuousmonitoring of PA signals from the ear blood microvessels revealed arapid appearance of Lymphazurin in the blood flow within a few minutesafter injection, followed by clearance of Lymphazurin from the bloodover the next 50 minutes.

The results of this experiment demonstrated that the prototype PAFCsystem exhibited sufficient sensitivity to detect the presence ofultrasonic contrast dyes in circulation.

Example 3. Detection of Nanoparticles Circulating in Rats by PrototypeIn Vivo Photoacoustic Flow Cytometry System

To demonstrate the sensitivity of the prototype in vivo photoacousticflow cytometry (PAFC) system described in Example 2, the followingexperiment was conducted. The prototype PAFC system was used to detectnanoparticles intravenously injected into the tail veins of rats.

The in vivo measurements in this experiment were performed using a ratmesentery model. The rat (White Fisher, F344) was anesthetized usingketamine/xylazine at a dosage of 60/15 mg/kg, and the mesentery wasexposed and placed on a heated microscope stage, and bathed in Ringer'ssolution at a temperature of 37° C. and a pH of 7.4. The mesenteryconsisted of transparent connective tissue of 7-15 μm thickness, and asingle layer of blood and lymph microvessels.

The nanoparticles used in this experiment were gold nanorods (GNRs),obtained from the Laboratory of Nanoscale Biosensors at the Institute ofBiochemistry and Physiology of Plants and Microorganisms in Saratov,Russia. On the basis of TEM and dynamic light scattering analyses, theGNR were estimated to be approximately 15 nm in diameter andapproximately 45 nm in length on average. The GNRs were used eitheruncoated, or the GNRs were functionalized using thiol-modifiedpolyethylene glycol (PEG) (Liao and Hafner 2005).

A 250-μL suspension of GNRs with a concentration of 1010 particles/mlwas injected into the tail veins of three rats, followed by thecontinuous monitoring of PA signals measured from 50-μm diameter bloodvessels in the rat mesentery using the PAFC system described in Example2. PAFC measurements were taken using a laser fluence of 100 mJ/cm², alaser beam diameter of approximately 50 μm, and a laser wavelength of830 nm, near the maximum absorption of the GNR.

Uncoated GNR were rapidly cleared from the blood circulation within 1-3minutes preferentially by the reticuloendothelial system (data notshown). After injection of the rats with PEGylated GNRs, strongfluctuating PA signals appeared with amplitudes significantly exceedingthe PA background signals from blood vessels within the first minute andcontinued for 14-25 minutes, depending on the individual animal. Inaddition, the PA background signal from the blood vessel increasedapproximately 1.5-2 times above the pre-injection background levels,reaching a maximum level between four and nine minutes after injection,as shown in FIG. 5 .

The averaged PA signals from the three rats, measured for 15 minutesafter injection with GNR suspensions, are summarized in FIG. 6 . Themaximum rate of individual PA signals per minute, indicative of thenumber of GNRs in circulation, was observed approximately 5 minutesafter injection, with a gradual decrease in the PA signal rate over thenext 10 minutes.

The results of this experiment demonstrated that the prototype PAFCsystem possessed sufficient spatial and temporal resolution tocontinuously monitor the circulation of nanoparticles as small as 15 nmin diameter. In addition, the prototype PAFC system was sufficientlysensitive to track fluctuations in the concentration of circulatingparticles from the time that they were injected to the time that theparticles were cleared from circulation.

Example 4. Detection of S. aureus Bacteria Circulating in Mice UsingPrototype In Vivo Photoacoustic Flow Cytometry System

To demonstrate the ability of the prototype photoacoustic flow cytometry(PAFC) system to detect bacteria cells in vivo under biologicalconditions, the following experiment was conducted. The prototype PAFCsystem, previously described in Example 2, was used to measure S. aureusbacteria circulating in nude mice.

The mouse ear model described in Example 2 was used the measurements ofcirculating bacteria in this experiment. Because the endogenous lightabsorption of S. aureus bacteria was relatively weak compared to theabsorption of other blood components in the NIR spectral range, thebacteria were labeled with the NIR-absorbing contrast substancesindocyanine green dye (ICG) and carbon nanotubes (CNT), due to theirrelatively high labeling efficiency and low toxicity (data not shown).

A S. aureus bacterium strain designated UAMS-1 was isolated from apatient with osteomyelitis at the McClellan Veterans Hospital in LittleRock, Ark., USA. The strain was deposited with the American Type CultureCollection and is available as strain ATCC 49230. UAMS-1 was cultured intryptic soy broth and grown aerobically for 16 h at 37° C. Cells wereharvested by centrifugation, resuspended in sterile PBS and incubatedwith Indocyanine Green (ICG) dye (Akorn Inc., USA) or carbon nanotubes(CNT) as described below.

Before incubation, ICG dye was filtered through a 0.22 μm pore sizefilter. A 150-μl aliquot of bacteria in suspension was incubated with375 μg of ICG in 150 μL of solution for 30 min at room temperature andthen for 2 h at 37° C. Labeled bacteria were centrifuged at 5,000 rpmfor 3 min and the resulting pellet was resuspended in PBS.

Single-walled carbon nanotubes (Carbon Nanotechnologies Inc., Houston,Tex., USA) and multi-walled carbon nanotubes (Nano-lab Inc., Newton,Mass., USA) used in this experiment were processed using known methods(Kim et al. 2006). The average length and diameter of the single-walledcarbon nanotubes were 186 nm and 1.7 nm respectively, and the averagelength and diameter of the multi-walled carbon nanotubes were 376 nm and19.0 nm respectively.

The carbon nanotube solutions were treated with five cycles of 1.5 minof ultrasound at a power of 3 W followed by 0.5 min of rest, for a totalof 10 minutes of interrupted ultrasound. A 150-μl aliquot of bacteria insuspension was incubated with 150 μL of CNT solution for 30 minutes atroom temperature followed by 2 additional hours of incubation at roomtemperature. Labeled bacteria were centrifuged at 10,000 rpm for 5 minand the resulting pellet was resuspended in PBS.

Labeled 100-μl suspensions of S. aureus bacteria at a concentration of5×10⁵ cells/ml were injected into each mouse's tail vein, and theclearance of the labeled bacteria was monitored using PAFC measurementstaken from 50-μm diameter microvessels in the ears of mice. Laser energywas delivered at a wavelength of 805 nm for the S. aureus that waslabeled with ICG, and at a wavelength of 850 nm for the S. aureus thatwas labeled with CNTs. For both label types, the laser energy wasdelivered at a beam diameter of approximately 50 μm and at a fluenceranging between 20 and 50 mJ/cm².

S. aureus bacteria labeled with ICG and CNT contrast substances yieldedcomparable results as summarized in FIG. 7 . After injection of labeledS. aureus, the prototype PAFC device detected a rapid appearance ofbacteria in the ear blood microvessels within the first minute, followedby a steady elimination of the bacteria from the blood circulation overthe next 3-5 minutes. Periodic PAFC monitoring of mice blood vesselsover the next few days revealed that very rare bacteria labeled with CNTor possibly unattached CNT continued to appear at an average rate of onePA signal every three minutes, and the labeled bacteria or CNT was notcompletely cleared from circulation until about 60 hrs after the initialinjection (data not shown).

The results of this experiment established the feasibility of PAFC forthe in vivo monitoring of individual cells in the circulatory systems ofliving organisms. Using appropriate contrast enhancement substances, thelaser fluence required for effective detection of cells in circulationwas well below the threshold levels for laser-induced cell damage.

Example 5. Detection of E. coli Bacteria Circulating in Mice UsingPrototype In Vivo Photoacoustic Flow Cytometry System

To demonstrate the ability of the prototype photoacoustic flow cytometry(PAFC) system to detect bacteria cells in vivo under biologicalconditions, the following experiment was conducted. The prototype PAFCsystem, previously described in Example 2, was used to detect the E.coli bacteria strain K12 in the circulation of nude mice.

The mouse ear model described in Example 2 was used for all measurementsof circulating bacteria in the experiments described below. Because theendogenous light absorption of E. coli bacteria was relatively weakcompared to the absorption of other blood components in the NIR spectralrange, the bacteria were labeled with NIR-absorbing carbon nanotube(CNT) markers.

The E. coli K12 strain was obtained from the American Type CultureCollection (Rockville, Md.) and maintained in Luria-Bertani (LB) mediumconsisting of 1% tryptone, 0.5% yeast extract, and 0.5% NaCl in aqueoussolution at a pH of 7. A 100-μl aliquot of E. coli in PBS was incubatedwith 100 μL of the CNT solution as described in Example 4 for 60 min atroom temperature.

100-μl suspensions of CNT-labeled E. coli bacteria at a concentration of5×10⁵ cells/ml were injected into each mouse's tail vein, and theclearance of the labeled bacteria was monitored using PAFC measurementstaken from 50-μm diameter microvessels in the ears of the mice. Laserenergy was delivered at a wavelength of 850 nm, a beam diameter ofapproximately 50 μm and at a laser fluence of 100 mJ/cm². PAFCmeasurements, summarized in FIG. 8 , detected a rapid appearance of thebacteria in circulation after injection, and the bacterialconcentrations in the blood decreased exponentially over the next 15-17minutes.

The results of this experiment confirmed the feasibility of PAFC for thein vivo monitoring of individual cells in the circulatory systems ofliving organisms. The laser fluence required for effective detection ofE. coli cells in circulation was well below threshold levels forlaser-induced cell damage.

Example 6. Detection of Circulating Exogenous Melanoma Cells UsingPrototype In Vivo Photoacoustic Flow Cytometry System

To demonstrate the ability to use the prototype in vivo PAFC device todetect unlabeled melanoma cells in circulation with extremely highsensitivity through skin cells with varying levels of melaninpigmentation, the following experiment was conducted.

B16F10 cultured mouse melanoma cells (ATCC, Rockville, Md.) weremaintained using standard procedures (Ara et al. 1990, Weight et al.2006, Zharov et al. 2006), including serial passage in phenol-free RPMI1640 medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetalbovine serum (FBS, Invitrogen). For comparison to the detection ofunlabelled melanoma cells, the endogenous NIR cell absorption wasincreased by staining with ICG (Akorn Inc., USA), a strongly absorbentdye in the NIR range, for 30 min at 37° C. and in the presence of 5%CO₂. No toxicity was observed after labeling as assessed using thetrypan blue exclusion assay (data not shown).

In vivo measurements of melanoma cells used the PAFC system previouslydescribed in Example 2 with a laser wavelength of 850 nm and a laserfluence of 80 mJ/cm². This wavelength was within a region in which theabsorbance of melanoma cells is relatively high compared to theabsorbance of hemoglobin, a major component of blood, as determined byin vitro measurements summarized in FIG. 9 .

To estimate the influence of endogenous skin melanin on PAFC detectionlimits, Harlan Sprague mice, strain NIH-BG-NU-XID were used in thisexperiment. Female mice of this strain possess high levels of skinpigmentation between 8 and 10 weeks of age. The mice were anaesthetizedand placed on a heated microscope stage as previously described inExample 2.

A 200-μl volume of saline solution containing approximately 10⁵ mousemelanoma cells was injected into the mouse circulatory system through atail vein and then monitored using the PAFC system. The number ofmelanoma cells per minute detected using PAFC for melanoma cells afterinjection are summarized in FIG. 10 for melanoma cells in mice with lowmelanin content (FIG. 10A) and for melanoma cells in mice with highmelanin content (FIG. 10B). In the first 5 minutes of PA detectionfollowing intravenous injection of cultured mouse melanoma cells,600±120 PA signals (representing melanoma cells) per hour were observed,and the rate of detection of melanoma cells steadily decreased over thesubsequent 20-30 min. Approximately 20 cells/hour and 4 cells/hour weredetected after 24 h and 48 h of monitoring, respectively. The initial PAsignal rate after the injection of melanoma cells stained with ICGcontrast enhancement substances was 720±105 cells/hour (data not shown).Assuming that all stained melanoma cells were detected by in vivo PAFC,82.4% of the unlabelled melanoma cells in circulation were detected byin vivo PAFC measurements.

The results of this experiment demonstrated the ability of the prototypein vivo PAFC device to detect and monitor the appearance and progressionof metastatic melanoma cells in circulation non-invasively.

Example 7. Detection of Circulating Spontaneous Metastatic Cells DuringTumor Progression Using the Prototype In Vivo PAFC Device

An experiment was conducted to determine the ability of the prototype invivo PAFC device to detect relatively scarce endogenous metastaticmelanoma cells circulating in lymph vessels. The PAFC system describedin Example 2 was used to monitor endogenous metastatic melanoma cells inmice. The laser characteristics used in this experiment are identical tothose described in Example 6.

Nude mice were anaesthetized and placed on the heated microscope stageas previously described in Example 2. The ear blood vessels underexamination were located 50-100 μm deep and had diameters of 35-50 μmwith blood velocities of 3-7 mm/sec. To increase the probability ofdetection of rare metastatic cells, blood vessels with relatively largediameters of 150-300 μm and flow velocities up to 10-30 mm/s in the skinof the abdominal wall were examined using a customized skin foldchamber.

50-μl suspensions containing 106 B16F10 cultured mouse melanoma cells(ATCC, Rockville, Md.) were subcutaneously injected into nude mice.Melanoma tumors subsequently formed in the ears of the mice and in theskin on the backs of the mice. PAFC was performed on ear and abdominalblood vessels to monitor the circulatory system for the appearance ofmetastatic cells. PA mapping was used to monitor the growth of tumors.

During ear tumor development, individual or groups of melanoma cellswere first detected in the skin area close to the tumor site on thesixth day following tumor inoculation using PA mapping measurements. PAmapping measurements utilized PA signals derived by scanning a focusedlaser beam with diameter of 10 μm across each mouse's ear. Metastaticcells first appeared in ear microvessels near the tumor on the twentiethday after inoculation at a rate of 12±5 cells/hour (data not shown).Surprisingly, during the same time period, no melanoma cells weredetected in the abdominal skin blood vessels. 25 days after inoculation,the average count of melanoma cells detected in the ear veins increasedto 55±15 cells/hour. At this same time, melanoma cells were detected inabdominal wall skin vessels at a rate of 120±32 cells/hour. Thirty daysafter inoculation, the detection rate decreased to 30±10 cells/hour inthe abdominal vessel, which may be attributed to inhibition ofmetastatic cell production in the primary tumor. PA mapping of selectedtissue and organs revealed multiple micrometastases in cervical andmesenteric lymph nodes, as well as in lung and liver tissues.

PAFC measurements of the nude mouse back tumor model revealed theappearance of metastatic melanoma cells in abdominal skin blood vesselsclose to the tumor site on day 5, much earlier than in the tumor earmodel. This indicates a much greater likelihood of detecting the initialmetastatic process in the vicinity of the primary tumor.

Thirty days after tumor inoculation, the average concentration ofmelanoma cells was 150±39 cells/ml, corresponding to a circulating rateof approximately 4-10 cells/min in a 50-mm blood vessel and a flowvelocity of 5 mm/s. The ultimate PAFC threshold sensitivity of the nudemouse back tumor model was estimated as 1 cell/ml. This circulating ratecorresponded to an incidence of approximately one melanoma cell among100 million normal blood cells.

The results of this experiment indicated that in vivo PAFC and PAmapping were sensitive methods with which to monitor the development ofmetastasized melanoma cells non-invasively, with high sensitivity andaccuracy.

Example 8. Detection of Spontaneous Metastatic Cells in LymphaticVessels During Tumor Progression Using the Prototype In Vivo PAFC Device

To determine the feasibility of detecting individual metastatic cells inlymph flow, the following experiment was conducted. A photoacoustic flowcytometer (PAFC) was used to monitor lymph flow for the presence of WBC,RBC, and metastatic melanoma cells.

The animal models used in this experiment were nu/nu nude mice, weighing20-25 g (Harlan Sprague-Dawley). PAFC measurements were obtained usingthe lymphatic vessels in the ears using a heated platform as describe\din Example 2. Melanoma tumors in the ear and back skin of the mice wereinduced by the subcutaneous injection of B16F10 mouse melanoma cells asdescribed in Example 6.

To locate the lymphatic vessels in the mouse ear, a PA mapping processusing a PA contrast agent was used. Ethylene blue (EB) dye, commonlyused for lymphatic research, was injected into the lymphatic vesselwalls. A 639 nm laser beam was then used to illuminate the lymphaticvessel, corresponding to the maximum absorption wavelength of EB dye,and the resulting PA signal emitted by the EB dye was monitored. Theposition of the laser beam on a lymph vessel was fixed when the PAsignal amplitude reached its maximum at the laser wavelength of 639 nm.

In vivo PAFC detection of unlabeled melanoma cells relied on melanin asan intrinsic cell marker, as in Example 7. Melanoma cells were detectedusing a laser wavelength of 850 nm, a laser fluence of 35 mJ/cm², and alaser beam diameter of approximately 50 μm. In mice with induced skinmelanomas, metastatic cells were observed to appear in a lymphaticvessel of the mouse's ear on the fifth day after inoculation at a rateof 1.2±0.5 cells/min, which steadily increased over the course of 2weeks (data not shown). In mice with a melanoma tumor in the ear,melanoma cells appeared in skin lymphatics 20 days after inoculation. 30days after inoculation strong PA signals detected the presence ofmetastatic melanoma cells in the sentinel lymph nodes, which was laterconfirmed by histology (data not shown). FIG. 11 shows the PA signalsdetected from single metastatic melanocytes circulating in the lymphaticvessel in the mouse ear five days after tumor inoculation.

The results of this experiment demonstrated the feasibility of detectingrelatively scarce metastatic melanoma cells circulating in the lymphaticsystem using in vivo PAFC techniques, with high sensitivity andaccuracy.

Example 9. Detection of Red Blood Cells and Lymphocytes SimultaneouslyCirculating in Lymph Vessels Using Prototype In Vivo PAFC Device

To determine the feasibility of detecting unlabeled individual cells ofdifferent types circulating in lymph flow, the following experiment wasconducted. A photoacoustic flow cytometer (PAFC) was used to monitorlymph flow for the presence of red blood cells and lymphocytes.

The animal models used in this experiment were 150-200 g rats (HarlanSprague-Dawley). PAFC measurements were taken using lymphatic vessels inthe mesentery of the rat, using the method described in Example 3.Lymphatic vessels were located, and the laser was focused on thelymphatic vessel using the methods described in Example 8.

Spectroscopic studies in vitro revealed that PA signals from lymphocytesreached maximal amplitude in the visible-spectral range near 550 nm,associated with cytochrome c acting as an intrinsic absorption marker(data not shown). Background PA signals from vessels and surroundingtissues were approximately 4-6-fold less than from single lymphocytes atthis wavelength due to the low level of background absorption and laserfocusing effects.

The prototype in vitro PAFC system described in Example 2 was used todetect circulating cells in the lymphatic vessels of the rat mesentery.The laser used in the PAFC system had a wavelength of 550 nm and afluence of 100 mJ/cm², and a circular beam diameter of approximately 50μm. The cell detection rate obtained in lymphatic vessels was 60±12cells/min. A graph showing the PA signals detected by the PAFC system ina rat mesentery lymphatic vessel is shown in FIG. 12 . Lymphocyteheterogeneity resulted in 2-2.5-fold fluctuations in PA signal amplitudefrom cell to cell. A small fraction of the detected cells had strong PAsignal amplitudes exceeding those of the lymphocyte signals by a factorof 10 to 20-fold. One such strong PA signal is shown as a white bar inFIG. 12 at 28 seconds. Subsequent spectral and imaging analysisidentified rare single red blood cells (RBCs) as the sources of theseexcessively strong PA signals.

The results of this experiment demonstrated that the in vivo PAFC systempossessed sufficient sensitivity and accuracy for the simultaneousdetection of red blood cells and lymphocytes circulating in thelymphatic vesicles.

Example 10. Detection and Identification of 3 Different ExogenouslyLabeled Cell Types in Circulation within Lymph Vessels Using a PrototypeIn Vivo Two-Wavelength PAFC Device

To demonstrate the ability of the photoacoustic flow cytometry (PAFC)system to detect cells using more than one wavelength of laser pulse,the following experiment was conducted. In this experiment, a PAFCsystem was used to detect exogenous blood cells that were labeled withthree different nanoparticles, while circulating in lymphatic vessels.The PAFC system detected the cells by illuminating the cells with laserpulses of two different wavelengths in the near-infrared (NIR) spectrum.

A PAFC system similar to that described in Example 2 was used to detectthe circulating cells. However, in the PAFC system used in thisexperiment, the laser of the PAFC system pulsed light at two differentwavelengths, corresponding to the wavelengths of maximum absorption fortwo of the nanoparticles used to label the cells. The first laser pulsewas at a wavelength of 865 nm, a laser fluence of 35 mJ/cm², and pulseduration of 8 ns. 10 μs after the end of the first laser pulse, a secondlaser pulse was delivered at a wavelength of 639 nm, a laser fluence of25 mJ/cm², and pulse duration of 12 ns. The paired laser pulses wererepeated at a frequency of 10 Hz.

The animal models used in this experiment were nu/nu nude mice, weighing20-25 g (Harlan Sprague-Dawley). PAFC measurements were taken usinglymphatic vessels in the mesentery of the mouse, using the methodsdescribed in Example 3. Lymphatic vessels were located, and the laserwas focused on the lymphatic vessel using the methods described inExample 8.

Normal fresh blood cells were obtained from heparinized whole-bloodsamples of donor mice after terminal blood collection. Red blood cellswere isolated by simple centrifugation, and lymphocytes were isolated byHistopaque (Sigma-Aldrich) density gradient centrifugation asrecommended by the supplier.

The nanoparticles used to label the various blood cells used in thisexperiment were gold nanorods (GNRs) and gold nanoshells (GNSs),provided by The Laboratory of Nanoscale Biosensors at the Institute ofBiochemistry and Physiology of Plants and Microorganisms in Saratov,Russia. The GNRs had an average diameter of 16 nm, an average length of40 nm, and a relatively narrow absorption wavelength band of 660±50 nm.The GNSs had an average diameter of 100 nm, and a maximum absorption ofwavelengths near 860 nm. Both the GNRs and GNSs were coated withpolyethylene glycol using the process described in Example 3.Single-walled carbon nanotubes (CNTs; Carbon Nanotechnologies Inc.) withan average length of 186 nm and an average diameter of 1.7 nm were alsoused as markers; the CNTs absorbed laser energy over a wide range ofwavelengths with an efficiency that monotonically decreases aswavelength increases (data not shown). All particles were in suspensionat a concentration of about 1010 nanoparticles/ml.

Live neutrophils were labeled with the GNSs, live necrotic lymphocyteswere labeled with the GNRs and apoptotic lymphocytes were labeled withthe CNTs. The cells were labeled by incubating 100-μl aliquots of eachcell type in phosphate-buffered saline with 100 μL of CNTs, GNRs, orGNSs for 15 min at room temperature.

The labeled cells, mixed in approximately equal proportions, wereintravenously injected into the tail vein of the mouse. About six hoursafter injection, mesenteric lymphatics were illuminated with two laserpulses at wavelengths of 865 nm and 639 nm as described above and usinga preparation similar to that described in Example 3. PA signals weredetected at a rate of 1-3 signals/min at this time.

The detected PA signals had one of three distinctive temporal shapesassociated with the response of the three different labels to the pairedlaser pulses, as shown in FIG. 13 . PA signals from necrotic lymphocytesmarked with GNR were generated in response to the 639 nm laser pulseonly, after a 10-μs delay, as shown in FIG. 13A. The apoptoticlymphocytes marked with GNS generated PA signals in response to laserpulse at a wavelength of 865 nm with no delay, as shown in FIG. 13B.Live neutrophils marked with CNT generated two PA signals after a 10-μsdelay, as shown in FIG. 13C; one PA signal was generated in response tothe 639 nm laser pulse, and the second PA signal was generated inresponse to the 850-nm laser pulse, due to comparable absorption by theCNT markers attached to the neutrophils at both wavelengths.

The results of this experiment demonstrated that labeling cells using avariety of contrast substances having different wavelength absorptioncharacteristics and illuminating the cells using two laser pulsewavelengths enabled the prototype in vivo PAFC device to detect anddiscriminate between live neutrophils, necrotic lymphocytes, andapoptotic lymphocytes circulating in the lymphatic vessels. This methodmay potentially be extended to unlabelled cells circulating in thelymphatic or circulatory systems, using two or more unique laser pulsewavelengths selected to generate a unique PA signal shape for each celltype to be detected.

Example 11. Spatial Resolution and Maximum Detectible Vessel Depth ofthe Prototype In Vivo PAFC System

To determine the maximum spatial resolution and maximum detectiblevessel depth of the prototype PAFC system, the following experiment wasconducted. The prototype PAFC system described in Experiment 2 and themouse ear model described in Example 7 were used to detect mousemelanoma cells injected into the tail veins of nude mice as described inExample 7.

The prototype PAFC system achieved a lateral resolution of 5-15 μm whendetecting melanoma cells circulating in mouse ear blood vessels withvessel diameters of 10-70 μm at depths of 50-150 μm below the surface ofthe skin. However, when melanoma cells circulating in mouse ear bloodvessels at a depth of 0.5 mm were measured, the lateral resolutiondecreased to 30-50 μm due to the scattering of the 850 nm laser pulsesby the additional tissue between the PAFC laser and the targeted bloodvessels.

The maximum depth at which the PAFC system was capable of detectingcells circulating in deep vessels was estimated by overlaying layers ofmouse skin of varying thickness over intact mouse skin containingperipheral blood vessels at a depth of approximately 0.3 mm below thesurface of the intact skin. Using the PAFC system with an unfocusedultrasound transducer (Panametrics model XMS-310, 10-MHz), PA signalswere detected at total skin thicknesses up to approximately 4 mm, with a27-fold signal attenuation due to light scattering. When a focusedultrasound transducer was used (Panametrics model V316-SM, 20 MHz, focallength 12.5 mm), PA signals were detected from melanoma cellscirculating in the mouse aorta at a depth of approximately 2.5 mm; alaser pulse wavelength of 850 nm was used to illuminate the melanomacells. At a total tissue depth as high as 11 mm, the PA signals emittedby circulating metastatic melanoma cells illuminated by 532 nm laserpulses remained discernible from the background PA signals emitted bysurrounding tissues. The lateral resolution at this vessel depth,measured by varying the angle of the ultrasonic transducer, wasestimated to be approximately 250 μm (data not shown).

The results of this experiment demonstrated that the PAFC system wascapable of detecting circulating melanoma cells at a vessel depth of upto 11 mm with a resolution of approximately 250 μm. This resolution maybe improved significantly through the use of higher frequency ultrasoundtransducers such as 50 MHz transducers.

Example 12. Assessment of the Spatial Resolution of a Prototype In VivoPAFC Device at Varying Skin Pigmentation Levels

To determine the sensitivity of the prototype PAFC system to the levelof skin pigmentation, the following experiment was conducted. The PAFCdevice described in Example 2 was used to measure PA signals from bloodvessels in nude mice skin with low and high levels of pigmentation usingmethods similar to those described in Example 7.

In the low-pigmented nude mouse model, the background PA signal fromskin cells was relatively weak. PA signals measured by a high frequencyultrasound transducer (Panametrics model V-316-SM, 20 MHz) resultingfrom the simultaneous irradiation of two circulatory vessels at depthsof approximately of 0.3 mm and 2.4 mm, were determined to have a timeseparation of approximately 1.4 ms. This delay is consistent withsignals emitted by cells with a separation distance of 2.1 mm, assuminga velocity of sound in soft tissue of approximately 1.5 mm/ms. Similarresults were obtained for measurements of circulatory vessels in thehighly pigmented nude mouse model (data not shown).

The results of this experiment demonstrated that the level of skinpigmentation did not significantly impact the spatial resolution of thePAFC device. For strongly pigmented skin, the assessment of deepervessels may be enhanced because the skin pigmentation may facilitate thediscrimination between PA signals from circulating individual cells andPA signals from the skin.

Example 13. Enrichment of Circulating Metastatic Cells in the Mouse EarModel

To determine the feasibility of novel methods for increasing theconcentrations of circulating metastatic cells detected by the in vivoPAFC device, the following experiment was conducted. Using the mouse earmodel to measure the incidence of circulating metastatic melanoma cells,as described in Example 7, the effect of gentle mechanical squeezing ofblood microvessels was assessed. This method of enriching the localincidence of rare circulating cancer cells in vivo exploited the sizedifferences between melanoma cells (16-20 mm), WBC (7-8 mm), and RBC(5-6 mm) and the high deformability of RBC compared to cancer cells. Thelumen size of the microvessel was decreased to 10-15 μm through gentlemechanical squeezing of blood microvessels in 50-μm microvessels ofmouse ear. After squeezing a 50-μm mouse ear blood vessel for 10 min,then quickly releasing the vessel, the rate of metastatic melanoma cellsmeasured by PAFC immediately after vessel release increasedapproximately 8-fold, relative to the rate measured before squeezing.The degree of blood vessel squeezing could be controlled by monitoringincreases and decreases in PA signal amplitudes.

The results of this experiment demonstrated that local enrichment ofcirculating metastatic melanoma cells was achieved through themechanical restriction of circulatory vessels.

Example 14. Manipulation of the Background Absorption by SurroundingBlood Cells Using Variations in Blood Oxygenation, Hematocrit, and BloodOsmolarity

To determine the effects of changes in blood oxygenation, hematocrit,and osmolarity on the background absorption of blood cells during invivo PAFC, the following experiment was conducted. The absorption oflaser energy by hemoglobin in its oxygenated (HbO₂) and deoxygenated(Hb) forms differs, depending on the oxygen saturation state of thehemoglobin and the wavelength of the laser pulse. The total absorptionof red blood cells decreases as oxygenation increases for laser pulsewavelengths 810-900 nm, and the absorption of red blood cells decreaseswith increasing blood oxygenation at laser pulse wavelengths of 650-780nm (data not shown). Thus, the oxygenation of the red blood cells may bemanipulated to reduce the background PA signals produced by red bloodcells.

Pure oxygen was delivered to a mouse using a mask around the mouse'shead, and the background PA signal obtained before and after theincreased blood oxygenation was measured using the in vivo PAFC systemdescribed in Example 2. The increased blood oxygenation resulting fromthe exposure of the mouse to pure oxygen for 15 minutes caused thebackground PA signal from veins to decrease by a factor of 1.36±0.14,using a laser pulse wavelength of 750 nm. Replacing the delivery of pureoxygen with the delivery of pure nitrogen led to a 35% decrease inbackground PA signal in an arteriole at a laser pulse wavelength of 900nm.

Another experiment was conducted to assess the effects of decreasing thedensity of the circulating RBC as measured by the hemotocrit on thebackground signal from circulating red blood cells. The hemotocrit of amouse's blood was temporarily reduced by the intravenous injection of0.5 ml of standard saline solution into the vein tail. After the salineinjection, PA signals from a 50-μm ear mouse vein dropped by a factor of2.3±0.3, and returned to near-initial levels within about 1.5 minutes.

Changes in blood osmolarity induced an increase in the RBC volume(swelling) that resulted in a decrease in the average intracellular Hbconcentration. Injection of 100-mL of hypertonic NaCl solution into themouse tail vein led to an approximately 2-fold decrease in the PA signalin the ear vein.

The results of these experiments demonstrated that the background PAsignals resulting from the emission of PA signals by red blood cells maybe reduced by manipulation of the chemical environment of the blood,including blood oxygenation, hemotocrit, and blood osmolarity. Theseapproaches may be readily applicable to human subjects because theprocedures used in this experiment are routinely performed in clinicalpractice.

Example 15. Assessment of Microbubbles Conjugated with Nanoparticles asPAFC Contrast Agents

To assess the effectiveness of microbubbles conjugated withnanoparticles as a contrast agent in PAFC, the following experiment wasconducted. Microbubbles (Definity Inc.) with average diameters of 2-4 μmwere incubated with PEG-coated gold nanoshells (GNS), previouslydescribed in Example 10, for 1 hr at room temperature. The measurementof PA signals in vitro, as described in Example 1, was conducted formicrobubbles only, for unconjugated GNSs, and for microbubblesconjugated with GNSs. The microbubbles conjugated with GNSs emitted thestrongest PA signals, the unconjugated GNSs emitted somewhat weaker PAsignals, and the microbubbles alone emitted negligible PA signals (datanot shown).

Increasing the energy of the laser pulses illuminating theGNS-conjugated microspheres led to a dramatic increase of the emitted PAsignals, followed by the disappearance of the microbubbles after asingle laser pulse. This observation was attributed to the laser-inducedoverheating of the GNSs leading to a dramatic temperature increase ofthe gas trapped inside of the microbubbles that ultimately ruptured themicrobubbles.

The results of this experiment demonstrated that microbubbles conjugatedwith GNS were an effective contrast agent, but that the energy of thelaser pulses may be constrained to avoid bursting the microbubbles.Because the microbubbles may be selectively attached to blood clots ortaken up by activated white blood cells, this contrast agent may expandthe potential applications of in vivo PAFC to include the detection ofblood clots and certain activated white blood cells.

Example 16. Detection of Circulating Exogenous Melanoma Cells Using thePrototype Two-Wavelength In Vivo PAFC Device

To demonstrate the ability to use two-wavelength in vivo PAFC to detectinjected unlabeled melanoma cells in circulation with extremely highsensitivity, the following experiment was conducted. B16F10 culturedmouse melanoma cells (ATCC, Rockville, Md.) were obtained and maintainedas described in Example 6. The experiments were performed using a nudemouse ear model similar, described in Example 2 (n=25). To mimicmetastatic cells, approximately 10⁵ tumor-derived B16F10 cells in a100-μl volume of saline solution were injected into the mousecirculatory system through a tail vein and then monitored in an ear veinusing an apparatus and methods similar to those described in Example 10.An ear blood vessel was illuminated by two laser pulses at wavelengthsof 865 nm and 639 nm with a 10-ms delay between the pulses.

The melanoma cells were distinguished from surrounding blood cells,based upon the distinctive absorption spectra of the melanoma cells, asdescribed previously in Example 6 and summarized in FIG. 9 . Melanomacells emitted two PA signals with a 10-ms delay in response to each pairof laser pulses. The first PA signal, induced by the 639 nm laser pulse,had a higher amplitude than the PA signal induced by the 865 nm pulse,as shown in FIG. 14A. Red blood cells, the most numerous blood cells incirculation, generated two PA signals with lower amplitudes than thecorresponding PA signals generated by the melanoma cells. In addition,for the red blood cells, the amplitude of the PA signal induced by the865 nm pulse was slightly higher than the PA signal induced by the 639nm laser pulse, as shown in FIG. 14B.

The PA signals corresponding to the melanoma cells were cleared over atwo-hour period following the injection, as shown in FIG. 15 .

Based on comparisons to similar data measured for melanoma cells labeledwith markers that emitted strong PA signals, it was estimated thatapproximately 89% of the unlabelled melanoma cells were detected (datanot shown). This percentage was lower than that found in previous invitro studies (96%) and indicated a false-negative-signal rate of 1.5cells/min related to the background signals of RBCs (data not shown).Longer-term monitoring of PA signals from ear blood vessels withoutprior melanoma cell injection detected no false-positive signals using asignal-to-noise ratio in excess of 2 as a false-positive criterion,where the signal noise was associated with fluctuations of laser energyproduced by the PAFC device and the density of red blood cells in thedetected volume.

The results of this experiment demonstrated that two-color in vivo PAFCwas an effective method for detecting metastatic melanoma cells incirculation. It was estimated that the method described above detectedapproximately 89% of the melanoma cells in circulation, with slightlylower detection rates due to skin pigmentation.

Example 17. Detection of Circulating Spontaneous Metastatic Cells DuringTumor Progression Using Two-Wavelength In Vivo PAFC

An experiment was conducted to determine the ability of two-wavelengthin vivo PAFC to detect relatively scarce endogenous metastatic melanomacells circulating in lymph vessels. The PAFC system described in Example10 was used to monitor endogenous metastatic melanoma cells in mice.Tumors were induced in nude mice by subcutaneous injections of melanomacells using methods similar to those described in Example 7. Tumorsformed and proliferated in the skin of the ears and the backs of thenude mice over a period of 4 weeks, as previously described in Example7.

PAFC was used to count spontaneous metastatic melanoma cells in anapproximately 50 μm-diameter ear blood vessel and in a 100-200μm-diameter skin blood vessel during tumor progression in the ear andskin of each mouse, as summarized in FIG. 16 . As previously describedin Example 7, the skin tumor growth rate was faster than that of the eartumors, and metastatic melanoma cells appeared more quickly in thecirculation, as indicated by the mean cell detection rate measured inthe skin capillaries, shown as solid square symbols in FIG. 16 . Inparticular, within the first week after the induction of the tumors,about 1-4 melanoma cells/min were detected in the skin vasculature, andas the tumor size increased, the rate of detection of metastaticmelanoma cells gradually increased to about 7 cells/min and about 12cells/min after 3 weeks and 4 weeks, respectively.

The results of this experiment indicated that two-wavelength in vivoPAFC was a sensitive method with which to monitor the development ofmetastasized melanoma cells non-invasively, with high sensitivity andaccuracy.

Example 18. In Vitro Photoacoustic Response of Quantum Dot Markers UsingTwo-Wavelength PAFC Device

An experiment was conducted to determine the ability of thetwo-wavelength PAFC device to detect quantum dot cell markers in vitro.The PAFC system described in Example 2 was used to measure photoacousticpulses emitted by quantum dots in response to laser pulses withwavelengths of 625 nm, pulse widths of 8 ns, and laser fluences ranging0.001-10 J/m². The laser beam used to pulse the quantum dots had adiameter of about 20-30 μm in the sample plane. Quantum dots wereobtained commercially with a polymer coating as well as with astreptavidin protein coating (Qdot 655 nanocrystals, Invitrogen,Carlsbad Calif.). The quantum dots had diameters of about 15-20 nm andan emission wavelength of about 655 nm. Either single quantum dots oraggregations of quantum dots were diluted with a buffer of 2% BSA/PBSand mounted on a microscope slide within a fluid layer of less than 1 μmdepth.

The two-wavelength PAFC system was used to pulse the quantum dotpreparation with laser fluences ranging from 0.001-30 J/m². Themagnitudes of the PA signals emitted by the quantum dots are summarizedin FIG. 17 . The quantum dot preparations had a non-linear PA signalresponse to the variations in laser fluences. PA signal amplitudegradually increased in the laser fluence range from 0.1-1 J/cm². Throughthe laser fluence range between 1.5-7 J/cm², the response increaseddramatically in a non-linear manner, and continued to increase inmagnitude up to a laser fluence of 15 J/cm². At laser fluences above 15J/m², the responses of the quantum dot preparations were saturated.

The PA signal response was assessed as a function of the number of laserpulses for laser fluences of 1.2, 4.0, 6.2, and 12.4 J/cm², assummarized in FIG. 18 . There PA signal was not significantly influencedby the number of laser pulses at laser fluences below about 3 J/cm²,indicating no blinking behavior, unlike the stereotypical fluorescentblinking behavior observed in quantum dots. At higher laser fluences,significant decreases in the PA signal amplitude were observed due to anincrease in the number of pulses, possibly due to laser induced meltingor thermal-based explosion of the quantum dots.

The results of this experiment indicated the quantum dots generatedstrong PA signals in response to laser pulses.

Example 19. Detection of Blood Clots In Vitro Using a NegativePhotoacoustic Contrast Detection Technique

To demonstrate the ability of the prototype PAFC system to detect clotsusing a negative photoacoustic contrast technique, the followingexperiments were conducted.

Whole blood samples were obtained from healthy human donors inheparinized tubes. Platelet-rich plasma was prepared via centrifugationof the whole blood at an acceleration of 200 g for 6 min to remove redblood cells (RBCs). Washed platelets were prepared via centrifugation ofplatelet-rich plasma an acceleration of 1,000 g in the presence ofprostacyclin (0.1 g/ml) for 10 min. The resulting pellet wasre-suspended in PBS to the desired concentration. The aggregations ofplatelets were initiated with collagen introduced into a 0.5 mL aliquotof platelet-rich plasma.

To determine the wavelength of maximum contrast between the plateletaggregations and surrounding blood cells, a conventional absorptionspectrum was obtained using a fiber spectrophotometer (USB4000, OceanOptics Inc, USA). Light transmitted at different wavelengths through aplatelet aggregation sample was collected through an ocular modifiedwith a custom-made fiber connector. The resulting absorption spectrum issummarized in FIG. 19. The platelet absorption spectrum is showncompared with spectra obtained in a similar manner using a whole bloodsample and using a suspension of gold nanorods. The maximum contrast ofthe platelets and the gold nanorods, defined in this experiment as themaximum difference between the PA signals generated by the platelets andthe gold nanorods, occurred between pulse wavelengths between about 600nm and about 750 nm.

The general layout of the PAFC system used for the detection of theplatelet aggregations in these experiments was assembled on thetechnical platform of an inverted microscope (IX81, Olympus America,Inc.). This platform incorporated photothermal (PT), photoacoustic (PA),fluorescent, and transmission digital microscope (TDM) modules, as wellas a tunable optical parametric oscillator (OPO, model Opolette HR 355LD, OPOTEK, Inc., Carlsbad, Calif.). The optical parametric oscillatorhad a spectral range of 410-2500 nm, a pulse width of 8 ns, a pulseenergy of up to 5 mJ, a pulse energy stability of 3-5%, a pulserepetition rate of 100 Hz, and a line width of about 0.5 nm. The pumplaser energy was controlled with a power meter (PE10-SH, OPHIR, Israel).The PAFC system was further equipped with two high pulse repetition ratelasers: a) a sapphire (Ti:S) laser (model LUCE 820-10 kHz, BrightSolutions, Inc., Italy) with a wavelength of 820 nm, a maximum pulseenergy of 78 μJ, a pulse width of 8 ns, and a pulse repetition rate of10 kHz, and b) a solid laser (model QL671-500, Crystal Laser, Reno,Nev., USA) with a wavelength of 671 nm, a maximum pulse energy of 40 μJ,a pulse width of 24 ns, and a pulse repetition rate of up to 50 kHz.

To conduct photothermal detection, a collinear probe beam from acontinuous wave stabilized He—Ne laser (Spectra model 117A; PhysicsInc., USA; wavelength=633 nm, power=1.4 mW) was used to inducetemperature-dependent variations of the refractive index aroundabsorbing targets, causing defocusing of the laser energy via thethermo-lens effect. The resulting PT signal from the irradiated volume(a reduction in the beam's intensity at its center) was detected by aphotodiode with a preamplifier (model PDA36A, ThorLabs Inc.) situatedbehind a pinhole. Photothermal imaging (PTI) in a scanning mode wasprovided by repositioning the samples to be imaged using a 3-dimensional(X-Y-Z) translation stage (model H117 ProScan II, Prior Scientific,Inc., USA) to an accuracy of 50 nm.

In addition, a customized photoacoustic (PA) module was included in thePAFC system that included an unfocused ultrasound transducer (model6528101, Imasonic Inc., Besancon, France) with a detection frequency ofup to 3.5 MHz and an amplifier (model 5660B, Panametrics-NDT, Olympus)with a maximum detection frequency of 5 MHz and a gain of 60 dB.Further, the PAFC system included a fluorescent imaging module (OlympusAmerica, Inc.).

The processing and storage of the recorded PT and PA data was performedusing a PC (model Precision 690 with a quadcore processor, 4 GB of RAMand Windows Vista 64-bit operating system, Dell Inc., Round Rock, Tex.)with a 200 MHz analog-to-digital converter digitizer board (modelPCI-5124, 12-bit, 128 MB memory, National Instruments, Inc., Austin,Tex.). Synchronization of the OPO operation and signal acquisition, aswell as control of the translation stage during scanning was implementedusing a proprietary software module (LabView 8.5, National Instruments,Inc., USA). Data signals were also recorded using a Tektronix TDS 3032Boscilloscope.

To determine whether sufficient contrast existed between the plateletaggregates and the surrounding blood cells, slides containing samples ofred blood cells, non-aggregated platelets, collagen-induced plateletaggregates in PBS, and collagen-induced platelet aggregates in wholeblood were visualized using optical imaging (FIGS. 21A-21D,respectively), photothermal detection (FIGS. 21E-21H, respectively), andphotoacoustic detection (FIGS. 21I-21L, respectively). For thephotothermal and photoacoustic detection, the samples were scanned usinga laser wavelength of 580 nm. Three different laser fluences were useddepending on the sample: 10 mJ/cm² for the RBC sample, 9.5 J/cm² for theplatelets in PBS, and 0.3 J/cm² for the platelet aggregation in theblood sample. The diameter of the laser spot used to illuminate thesamples was 7±2 μm. Referring to FIG. 21L, the photoacoustic detectionof a platelet aggregate surrounded by red blood cells was characterizedby negative contrast, in which the amplitude of the photoacoustic (PA)signal produced by the platelet aggregate decreased to levelssignificantly less than the PA signal amplitudes produced by thesurrounding red blood cells. Based on the distinct negative contrast ofthe PA signal produced by the platelet aggregation relative to the PAsignal amplitude produced by the surrounding red blood cells, theplatelet aggregates were identifiable based on PA imaging.

To determine if PA imaging could be used to determine the spatial extentof a platelet aggregate, a collagen-induced platelet aggregate with asize of about 20 μm was placed in a blood sample with a sample thicknessof about 120 μm on a glass slide. The sample was scanned using PAscanning at a laser wavelength of 532 nm, a pulse repetition frequencyof 100 Hz, and a pulse energy of 1.0 μJ. The diameter of the laser spotwas about 5 μm and the scan was performed using translation intervals of10 μm per step. The scan was repeated eight times and the results wereaveraged. The results of the photoacoustic scanning are summarized inFIG. 22 . The PA signal obtained from the scan of a platelet aggregatewith the size of about 20 μm exhibited a significant negative contrastrelative to the background PA signals from the surrounding blood cells.

To assess the variation of the magnitude of the negative contrast ofplatelet aggregates as a function of the size of the aggregate,collagen-induced platelet aggregates with sizes ranging from about 10 μmto about 120 μm were placed in blood samples with a sample thickness ofabout 120 μm on glass slides and subjected to PT and PA imaging. FIG. 23is a summary of the negative contrast of the collagen-inducedaggregates, expressed as a percent reduction of the PA signal relativeto the baseline signal levels from the surrounding blood cells. Assummarized in FIG. 23 , the PA and PT signal amplitudes were dependenton the size of the aggregates. The negative contrast resulted in signallevels that were discernable from the background signal from thesurrounding red blood cells for aggregates as small as 20 μm.

The results of this experiment demonstrated that platelet aggregateswere detectable within the background signal produced by surroundingblood cells using the negative contrast of the PA and PT signalsproduced by the platelets relative to the corresponding signals producedby the surrounding blood cells. Further, the magnitude of the negativecontrast was related to the size of the platelet aggregate.

Example 20. Detection of the Structure of Heterogeneous Blood Clots InVitro Using the PAFC System

To demonstrate the ability of the PAFC system to detect the structure ofheterogeneous blood clots, the following experiments were conducted.

Blood samples were obtained, and collagen was added to the blood samplesto induce the formation of a heterogeneous clot, as described in Example19. Optical images and PT/PA scans of the clot were obtained using theequipment and methods described in Example 19.

FIG. 24A is an optical image of the heterogeneous blood clot formed in awhole blood sample, and FIG. 24B is the corresponding photoacousticsignal pattern from the PA scan of the sample. As shown in FIG. 24B, therelatively high density of red blood cells in the outer layer of theclot was characterized by a distinct increase in the magnitude of the PAsignal above background levels (positive contrast), and the relativelyhigh density of platelets in the center of the clot is characterized bya distinct decrease in the magnitude of the PA signal below backgroundlevels (negative contrast).

The results of this experiment demonstrated the feasibility ofidentifying the structure of aggregations of red blood cells andplatelets using the PAFC system.

Example 21. In Vivo Detection of Circulating Platelet-Rich Clots in RatMesentery Model Using PAFC

To assess the feasibility of detecting circulating clots in vivo usingthe PAFC system, the following experiments were conducted. Photoacousticand photothermal measurements were conducted in vivo using circulatoryvessels in the mesentery of the rat, using a method similar to Example 3and the PAFC system described in Example 19. The formation of clots inthe rats was induced using an intravenous injection of collagen (No. 385Collagen, Chrono-Log) at a dosage of about 100 μg/kg of body mass.

Intravenous injection of collagen into the rats (N=3) led to theformation of circulating platelet-rich clots within a few minutes asshown in FIG. 25 . FIG. 25A is an optical image of normal blood flowbefore the formation of a clot (control), and FIG. 25B is an opticalimage of a clot adhered to the epithelial wall of a mesentery bloodvessel. The platelet-rich clots were identified using PAFC; the resultsare summarized in FIG. 26 . FIG. 26A and FIG. 26B are representativephotothermal and photoacoustic signal patterns, respectively, detectedfrom blood flow prior to the formation of clots. FIG. 26C and FIG. 26Dare representative photothermal and photoacoustic signal patterns,respectively, detected from a circulating clot. The detection of thecirculating clot was further verified by comparison to high-speedimaging taken concurrently with the PAFC measurements.

The PA signals from circulating clots were characterized by negativedips in signal magnitude corresponding to a negative contrast. Themagnitude of the negative contrast, the duration of the negative dips,and the rate of clot signal detection varied within large ranges of20%-100%, 10-100 ms and 0.5-3.0 clot/min, respectively, which indicatedconsiderable heterogeneity in clot size, velocity, and concentration(N=6). Some clots were observed to be adhered to the vascular wall about10-15 min after the collagen injection, as shown in FIG. 25B.

Initially, the adhered clots appeared to be almost transparentstructures with no sign of incorporated RBCs. After about 5 minutes ofadhesion, single RBCs were incorporated into the clots. The number ofRBCs incorporated into the clots then gradually increased over the next15 minutes of adhesion.

These observations suggested that newly-formed clots circulated asplatelet-rich white clots at similar velocities as surrounding RBCs,resulting in minimal collisions and minimal incorporation of RBCs intothe circulating white clots. By contrast, the rate of collisions ofmoving RBCs with static adhered clots increased dramatically, leading toRBCs being stuck in the heterogeneous meshes formed by platelets andfibrin fibers. Although the transformation of white clots into partlyred clots led to a decrease of negative contrast in PAFC measurements,the contrast remained sufficiently high for clot detection.

The results of this experiment demonstrated that clots of variouscompositions were detectable using PAFC visualization methods.

Example 22. In Vivo Detection of Circulating Platelet-Rich Clots in theMouse Ear Model Using PAFC

To assess the efficacy of in vivo detection of collagen-induced clots ina mouse ear model, the following experiments were conducted. PAFCmeasurements were conducted in vivo to detect clots circulating throughcapillaries in the ear of a mouse, using methods similar to thosedescribed in Example 2 and the PAFC system described in Example 19. Theformation of clots in the mice was induced using an intravenousinjection of collagen (No. 385 Collagen, Chrono-Log) at a dosage ofabout 100 μg/kg of body mass.

After collagen injection, real-time monitoring of the collagen-inducedcirculating clots was performed in each mouse ear (N=3) using PAFC.Because transmission imaging had low resolution due to strong lightscattering in the mouse ear tissue, the presence of circulating clotsdetected by PAFC was verified by labeling the circulating clots withconventional fluorescent dye (CFSE) and performing fluorescentmicroscopic visualization. Ultra-fast PAFC performed using a highpulse-repetition rate (10 kHz) laser at an pulse wavelength of 532 nmdetected circulating clots associated with negative PA signal dips at adetection rate of 1-2 clots/min and negative contrast levels rangingfrom 12% to 59% of the mean background PA signal levels, as shown inFIG. 27 . A thromboembolism was later found in the lungs of the mice,consistent with previously published studies of collagen-induced clots.

The results of this experiment demonstrated that blood clots circulatingthrough capillaries beneath pigmented mouse skin were detectable in vivousing the PAFC system.

Example 23. In Vivo Detection of Circulating Clots in a Mouse CarotidArtery Using PAFC

To assess the efficacy of in vivo detection of clots circulating in amouse carotid artery, the following experiments were conducted. PAFCmeasurements were conducted in vivo to detect clots circulating throughthe carotid arteries of nude mice similar to those described in Example2 and using the PAFC system described in Example 19. The formation ofclots in the mice (N=3) was induced using an intravenous injection offerric chloride, which caused collagen release and platelet activationfollowed by the formation of initially platelet-rich white clots thattransformed over time into heterogeneous red thrombi. A gradual decreasein PA signal variation from the carotid arteries was detected over thefirst 5 minutes following the injection of the ferric chloride. Thisdecrease was associated with clot formation and accompanied by changesin the shape and the width of PA signals. FIG. 28A shows arepresentative PA signal taken from normal blood flow through thecarotid artery prior to injection, and FIG. 28B shows a representativePA signal taken from the carotid artery after total occlusion of bloodflow by a clot. Over time rare transient PA signals with a specific setof positive and negative contrasts were found in circulation, that werelikely related to detached fragments of the carotid thrombus circulatingas moving clots. FIG. 28C shows a PA signal from a circulating clotdetected in a skin circulatory vessel of the mouse.

The results of this experiment demonstrated that blood clots formingwithin a carotid artery were detectable in vivo using the PAFC system.

Example 24. In Vivo Detection of Circulating Clots in Presence ofCirculating Tumor Cells Using Two-Color PAFC

To assess the in vivo detection of collagen-induced clots in thepresence of circulating tumor cells (CTCs) in a simulated flow, thefollowing experiments were conducted. PAFC measurements were conductedto monitor the blood circulating through a mouse ear vessel using PAFCmethods similar to those described in Example 22.

Breast tumor cells (MDA-MD-231) were labeled by gold nanorods having amaximum absorption near 670 nm, using methods similar to those describedin Example 10. The labeled breast tumor cells were intravenouslyinjected into the mice. The formation of clots in the blood flow of themice was induced by intravenous injection of collagen into the mice(N=6).

Previous in vitro observation of the formation of clots in mouse orhuman blood spiked with tumor cells in an artificial flow device (notshown) exhibited the rapid formation of clots within a short time (5±1.5min) after collagen injection. The clots were observed to be aggregatesof platelets, microparticles, tumor cells and leukocytes that varied incomposition and size. Unexpectedly, about 20-30% of the clots containedCD44 markers associated with the most aggressive population of cancerstem cells (data not shown). Approximately 10% of these clots includedCD45 marker associated with leukocytes (data not shown).

After the injection of collagen, two-color PAFC conducted using laserpulses at 532 nm and 671 nm was used to monitor the blood flow through amouse ear vessel, using a two-color PAFC method similar to the methoddescribed previously in Example 10. A representative sample of thephotoacoustic signals detected by the PAFC system induced by 532 nm and671 nm excitatory pulses is presented in FIG. 29 . The PA signalsgenerated by the 532 nm excitatory pulse exhibited several sharp dropsin signal magnitude that were associated with the detection of clotscontaining platelets. However, several unique patterns of PA signalswere detected during PA monitoring that were associated with particularcirculating structures.

Examples of these PA signal patterns are highlighted as inset graphs inFIG. 29 . A negative contrast in the PA signal produced by the 532 nmpulse accompanied by no change in the PA signal produced by the 671 nmpulse indicated the detection of a clot that included mostly platelets(left inset). Negative contrast at 532 nm detected simultaneously with apositive contrast in the PA signal produced by the 671 nm pulseindicated the detection of a CTC-platelet clot complex (center inset).The detection of positive contrast at 671 nm without a simultaneousnegative contrast at 532 nm was associated with the detection ofcirculating tumor cells not associated with clots (right inset).

The results of these experiments indicated that high resolutionmulticolor PAFC may be used to distinguish the composition, size, andconcentration of clots in vivo by analyzing PA signal shapes, widths,and rates of signal detection, respectively.

Example 25. Signal Processing of PA Signals from Clots Detected UsingPAFC

To assess the effect of signal processing techniques such as frequencyfiltering and data averaging on the PA signal patterns used to detectclots in circulation, the following experiments were conducted.

PA signals were obtained in vivo from circulating mouse blood containingmoving clots, using methods similar to those described in Example 22.The PA signal was processed through a high pass filter with a cutofffrequency of 100 Hz. The effect of frequency filtering the PA signaldata through the 100 Hz high pass filter is illustrated in FIG. 30 .FIG. 30A is a graph showing a representative sample of the pre-filteredPA signals recorded over a three-minute period. The pre-filtered signalsexhibit oscillations having a variety of characteristic frequencies.Superimposed on this oscillating signal are two pronounced negative PAsignal dips produced by clots at recording times of about 680 secondsand 760 seconds. Much of the lower-frequency oscillations observedwithin this recorded PA signal are likely artifacts introduced bymovements of the animal's blood vessel due to the animal's pulse orbreathing. FIG. 30B is a graph showing the same series of PA signalsafter processing through the 100 Hz high pass filter. The high passfilter eliminated the lower frequency oscillations below 100 Hz withinthe data, but retained the pronounced negative dips associated with thedetection of clots at both recording times. The PA negative contrastdips consisted predominantly of high frequency signal components in therange of 100 kHz to about 5 MHz.

To assess the effect of data averaging, recorded PA signal data weresubjected to a 100 Hz high pass filter to minimize the lower frequencyoscillations as described above. The filtered PA signals produced fromconsecutive pulses of the 10 kHz laser of the PAFC were averaged inincreasingly large groups ranging from no averaging (N=0) to averagingthe PA signals of 1024 consecutive laser pulses (N=1024).

FIG. 31 summarizes the effect of signal averaging on a PA signal patternthat included a negative contrast dip resulting from the detection of ablood clot. Because a high laser pulse rate of 10 MHz was used to obtainthese data, the signal-to-noise ratio of the PA signals was much higherdue to the detection of multiple PA signals from the same bloodcomponents induced by multiple consecutive laser pulses. As shown inFIG. 31A, signal noise overwhelms the negative contrast dip detected atabout 757.5 seconds. Signal averaging over 16 PA signals (FIG. 31B), 64PA signals (FIG. 31C), 256 PA signals (FIG. 31D), and 1024 PA signals(FIG. 31E) reduced the signal-to-noise ratio, and enhanced the detectionof the negative contrast dip at 757.5 seconds.

The results of this experiment demonstrated that signal processing of PAsignal data using methods such as high pass filtering and data averagingresulted in enhanced detection of negative contrast dips and enhancedthe sensitivity of the PAFC system for the detection of circulatingblood clots.

It should be understood from the foregoing that, while particularembodiments have been illustrated and described, various modificationscan be made thereto without departing from the spirit and scope of theinvention as will be apparent to those skilled in the art. Such changesand modifications are within the scope and teachings of this inventionas defined in the claims appended hereto.

REFERENCES

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1-21. (canceled)
 22. A method for continuous monitoring of a circulatoryvessel of a living organism, the method comprising: pulsing circulatingtarget objects comprising red blood cells and at least one clot withinthe circulatory vessel with at least one pulse of laser energy at afirst pulse wavelength ranging between 400 nm and 2500 nm, wherein thefirst pulse wavelength induces a photoacoustic signal from thecirculating target objects, wherein the at least one clot is ahemoglobin-rich red clot, a platelet-rich white clot, or a combinationthereof and wherein the hemoglobin-rich clot is light absorbing and theplatelet-rich white clot is non-absorbing; obtaining a photoacousticpattern induced by the at least one pulse of laser energy, wherein thephotoacoustic pattern comprises at least one photoacoustic signalcomprising a blood background signal produced by the red blood cells;and simultaneously obtaining fluorescence, scattering, and photothermalsignals from the target objects, via a photodetector.
 23. The method ofclaim 22, the method further comprising: processing photoacoustic,fluorescence, scattering, and photothermal signals; analyzing acombination of the photoacoustic pattern and the fluorescence,scattering, and/or photothermal signals to determine the presence of theat least one clot, wherein analyzing the photoacoustic pattern comprisesdetermining the presence of positive and negative contrast peaks in thephotoacoustic pattern, wherein the negative contrast in thephotoacoustic pattern below the blood background signal indicates thepresence of the white clot, the positive contrast in the photoacousticpattern above the blood background signal indicates the presence of thered clot, and a combination of positive contrast and negative contrastindicates the presence of a combination of red and white clots; andproducing a detection signal when the photoacoustic pattern and thefluorescence signal, scattering signal, photothermal signal, orcombinations thereof indicates the presence of a clot.
 24. The method ofclaim 23, wherein processing photoacoustic, fluorescence, scattering,and photothermal signals comprises signal filtering and signal averagingto eliminate signal fluctuations due to breathing, blood pulse, or otherbody movements or signal noise using a high-pass filter, a low-passfilter, and combinations thereof.
 25. The method of claim 23, whereinanalyzing the combination of the photoacoustic pattern and thefluorescence includes analyzing at two wavelengths photoacoustic signalshapes, widths, and rates of signal detection to distinguish acomposition, size, and concentration of clots.
 26. The method of claim22, wherein the circulatory vessel includes capillaries, arterioles,venules, arteries, veins including a carotid artery and lymphaticvessels with diameters ranged between about 10 μm and about 2 cm. 27.The method of claim 26, wherein the circulatory vessel is located invarious organs and tissues, including, but not limited to skin, lips,eyelid, interdigital membrane, retina, ear, nail pad, scrotum, brain,breast, prostate, lung, colon, spleen, liver, kidney, pancreas, heart,testicles, ovaries, lungs, uterus, skeletal muscle, smooth muscle, andbladder.
 28. The method of claim 22, the method further comprising:simultaneously irradiating circulatory vessels at different depths;measuring photoacoustic signals from blood vessels at different depthsby determining a signal delay; and determining the depths of the bloodvessels based on the signal delay.
 29. The method of claim 23, themethod further comprising: determining the presence and extent of the atleast one clot; diagnosing a stroke, heart attack, or other clot-relateddisorders based on the presence and extent of the at least one clot; anddirecting an application of an anti-clot treatment.
 30. The method ofclaim 23, the method further comprising issuing an alarm after receivinga detection signal indicating an alarm condition; wherein the alarmcondition is a detection signal of sufficient magnitude or two or moredetection signals at sufficiently high frequency, and wherein the alarmis a continuous or intermittent visual display, audible sound,vibration, or signal to summon medical assistance.
 31. The method ofclaim 29, the method further comprising: sending a communication to ahospital, medical center, or medical assistance personal to summonmedical assistance, wherein the communication is sent by Bluetoothsignals, cellular signals, or wireless Internet signals; andadministering an anti-clotting treatment based on a processed signalquantity of the detection signal, wherein the processed signal quantityis signal magnitude, frequency of signals, or elapsed time since aprevious detection signal, and wherein the anti-clotting treatment is ananti-clotting medication administered continuously or in discrete dosesor pulsing of the clot with a high-intensity laser pulse at a laserfluence sufficient to eliminate the clot.
 32. The method of claim 22,wherein the target objects are unlabeled biological cells, platelets,red blood cells, live, apoptotic and necrotic white blood cells, unboundcontrast agents, biological cells labeled using contrast agents,aggregations of cells, sickle cells, infected cells, inflamed cells,stem cells, dendritic cells, metastatic cancer cells resulting frommelanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, andtesticular cancer, bacteria, viruses, fungal cells, protozoa,microorganisms, pathogens, animal cells, plant cells, and leukocytesactivated by various antigens during an inflammatory reaction,heterogeneous clots comprising platelets, products resulting from cellmetabolism or apoptosis, cytokines or chemokines associated with theresponse of immune system cells to infection, exotoxins and endotoxinsproduced during infections, specific gene markers of cells such astyrosinase mRNA and p97 associated with cancer cells, MelanA/Mart1produced by melanoma cells, PSA produced by prostate cancer, andcytokeratins produced by breast carcinoma, and any combination thereof.33. The method of claim 23, wherein the detection of the circulatingclot is verified by comparison of high-speed optical imaging takenconcurrently with the photoacoustic pattern.
 34. The method of claim 23,further comprising reducing background photoacoustic signals by:delivering oxygen or nitrogen to reduce changes in blood oxygenation;injecting standard saline solution to decrease hematocrit; and injectinghypertonic NaCl solution to reduce changes in blood osmolarity.
 35. Themethod of claim 22, further comprising enriching circulating metastaticcells and clots by mechanically squeezing micro blood and lymph vessels.36. The method of claim 32, wherein the contrast agents are chosen fromthe group including indocyanine green dye, melanin, fluorosceinisothiocyanate (FITC) dye, Evans blue dye, Lymphazurin dye, trypan bluedye, methylene blue dye, propidium iodide, Annexin, Oregon Green, C3,Cy5, Cy7, Neutral Red dye, phenol red dye, AlexaFluor dye, Texas reddye, gold nanospheres, gold nanoshells, gold nanorods, gold cages,carbon nanoparticles, prefluorocarbon particles, carbon nanotubes,carbon nanohorns, magnetic nanoparticles, quantum dots, binarygold-carbon nanotube nanoparticles, multilayer nanoparticles, clusterednanoparticles, liposomes, liposomes loaded with contrast dyes, liposomesloaded with nanoparticles, micelles, micelles loaded with contrast dyes,micelles loaded with nanoparticles, microbubbles, microbubbles loadedwith contrast dyes, microbubbles loaded with nanoparticles, dendrimers,aquasomes, lipopolyplexes, nanoemulsions, polymeric nanoparticles, andcombinations thereof.
 37. The method of claim 32, wherein contrastagents are conjugates with antibodies and other ligands to targetobjects including CD45 as a marker associated with leukocytes and CD44as a marker of cancer stem cells.
 38. The method of claim 23, the methodfurther comprising: incubating at least one microbubble with an averagediameter of 2-4 μm with PEG-coated gold nanoshells (GNSs); attaching theat least one microbubble to a blood clot or activated white blood cell;and increasing the laser energy of the laser pulses illuminating the atleast one microbubble to increase emitted photoacoustic signals.
 39. Adevice for continuous monitoring of a circulatory vessel of a livingorganism, comprising: an in vivo flow cytometer, wherein the in vivoflow cytometer comprises: a pulsed laser configured to pulse targetobjects comprising light absorbing at least one hemoglobin rich red clotand/or at least one non-absorbing platelet-rich white clot within thecirculatory vessel with a plurality of laser pulses at a pulsewavelength ranging between about 400 nm and about 2500 nm; an opticalmodule configured to convert the wavelength, pulse rate, or bothwavelength and pulse rate of the laser pulses emitted by the pulsedlaser to desired values; an ultrasound transducer configured to receivephotoacoustic signals emitted by the target objects in response to thelaser pulses and generate an output; and a photodetector configured toreceive fluorescence, scattering, and/or photothermal signals emitted bythe target objects.
 40. The device of claim 39, wherein the devicefurther comprises a processor, the processor comprising: an amplifier; adata recording system; and a data analysis system having data analysissoftware, wherein the processor is configured to: receive the outputfrom the ultrasound transducer and the fluorescence, scattering, and/orphotothermal signals from the photodetector, wherein the outputcomprises a photoacoustic pattern; and analyze a combination of thephotoacoustic pattern, the fluorescence signal, the scattering signal,and/or the photothermal signal to determine the presence of the at leastone red clot, wherein analyzing the photoacoustic pattern comprisesdetermining the presence of positive and negative contrast peaks in thephotoacoustic pattern, wherein the negative contrast in thephotoacoustic pattern below a background photoacoustic signal indicatesthe presence of the white clot, the positive contrast in thephotoacoustic pattern above the background photoacoustic signalindicates the presence of the red clot, and a combination of positivecontrast and negative contrast indicates the presence of a combinationof red and white clots.
 41. A self-contained wearable device forcontinuous monitoring of a circulatory vessel of a living organism,comprising: a miniaturized in vivo flow cytometer; a clot monitoringsystem; an alarm system to provide a warning to the living organismand/or summon medical attention; and a power source, wherein theminiaturized in vivo flow cytometer, the clot monitoring system, thealarm system, and the power source are removably attached to anappendage of the living organism using straps, adhesive patches, oradhesive strips, and wherein the appendage is chosen from a neck, wrist,finger, forearm, upper arm, hand, foot, ankle, lip, ear, chest, abdomen,eye head, scalp, or leg.